Lysine conjugated immunoglobulins

ABSTRACT

Provided herein are conjugated immunoglobulins and methods for generating conjugated immunoglobulins using a microbial transglutaminase.

RELATED APPLICATIONS

This application is a continuation application of International Application PCT/JP2017/021672, filed on Jun. 12, 2017, which claims priority to U.S. Provisional Patent Application No. 62/348,410, filed on Jun. 10, 2016. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 30, 2017, is named 118557-03902_Sequence_Listing.txt and is 638,976 bytes in size.

TECHNICAL FIELD

Provided herein are lysine conjugated immunoglobulins and methods of creating the same.

BACKGROUND

The utility of monoclonal antibodies extends from basic research to therapeutic and diagnostic applications. The ability to conjugate antibodies to functional agents extends their functionality even further. The manufacture of conjugated antibodies usually involves conjugation of a linker, drug, or other functional agent to reactive lysine cysteine residues on the heavy (HC) and light (LC) chains of a monoclonal antibody (mAb). See Deonarain, et al., “Emerging formats for next-generation antibody drug conjugates”, Expert Opinion in Drug Discovery (2015), 10(5): 463-481. Lysine conjugation is typically mediated by succinimide (NHS)-based or isothiocyanate-based chemistry. Cysteine-based conjugation requires partial reduction of the antibody to break some of the interchain disulfide bonds, thereby creating free thiol side chains. Thiol-reactive functional agents can then react with the free thiol groups on the antibody to generate antibody-drug conjugates (ADCs). Both of these methods result in modification of multiple lysines or cysteines leading to heterogeneous mixtures of ADCs with a distribution of drug-to-antibody (DAR) ratios and drug modifications at random positions.

A recent push to utilize site-specific conjugation technologies as a way to produce a homogeneous ADC product with a defined DAR has yielded several methods including engineering unpaired cysteines, incorporation of non-natural amino acids, and site-specific enzymatic modification. While these methods produce homogeneous products, they each have their disadvantages. Cysteine-based conjugation requires an added step to remove a capping cysteine, glutathione, or even a light chain from the unpaired cysteine. See, e.g., Junutula, et al., “Site-Specific Conjugation of a Cytotoxic Drug to an Antibody Improves Therapeutic Index”, Nature Biotechnology, (2008) 26:925-932; Chen, et al., “Charge-based Analysis of Antibodies with Engineered Cysteines”, MAbs (2009) 1(6): 563-571; Gomez, et al., “Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture” Biotechnol Progress (2010), 26: 1438-1445. Further, serum instability of maleimide-based chemistry currently used for cysteine-based conjugates has been demonstrated raising concerns for loss in potency or off-target toxicity. Alley, et al., “Contribution of Linker Stability to the Activities of Anticancer Immunoconjugates”, Bioconjugate Chemistry (2008) 19(3): 759-765; Shen, et al., “Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates”, Nature Biotechnology (2012) 30: 184-189. Incorporation of non-natural amino acids requires expression in either a genetically modified cell-based or cell-free system. Hallam, et al., “Unnatural Amino Acids in Novel Antibody Conjugates”, Future Med. Chem. (2014) 6(11): 1309-1324. Further, the presence of an unnatural amino acid could trigger an immunogenic response in patients. Site-specific enzymatic modifications, however, could potentially utilize a native, wild-type amino acid in the antibody sequence, thereby minimizing the chance for immunogenicity. Further, the post-translational bonds typically formed by protein-modifying enzymes are very stable.

Site-specific enzymatic modification of proteins has been explored using a family of proteins called transglutaminases that catalyze the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ε-amino group (acyl acceptor) of a lysine (see FIG. 1) (see, e.g., Yokoyama, et al., “Properties and Applications of Microbial Transglutaminase”, Appl. Microbiol. Biotech. (2004) 64: 47-454; Strop, “Versatility of Microbial Transglutaminase”, Bioconjugate Chemistry, (2014) 25(5): 855-862; Kieliszek et al., “Microbial Transglutaminase and its Application in the Food Industry”, Folia Microbiol (2014) 59:241-250). Recently, several groups have explored utilizing transglutaminase as a means to produce ADCs (see, e.g., Josten et al., “Use of Microbial Transglutaminase for the Enzymatic Biotinylation of Antibodies”, J Immunol Methods, (2000) 240:47-54; Mindt et al., “Modification of Different IgG1 Antibodies via Glutamine and Lysine Using Bacterial and Human Tissue Transglutaminase”, Bioconjugate Chemistry (2008) 19(1): 271-278); Jeger, et al., “Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase” Angew. Chem. Int. Ed. Engl. (2010) 49: 9995-9997; Strop et al., “Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates”, Chem Biol (2013) 20(2):161-167; Dennler et al., “Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody—Drug Conjugates”, Bioconjugate Chemistry (2014) 25(3): 569-578; Siegmund, et al., “Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation”, Angew. Chem. Int. Ed. Engl. (2015) 54(45):13420-13424). Transglutaminases are found in organisms ranging from bacteria through humans that are structurally and functionally related, yet each is involved in specific cellular processes. A microbial transglutaminase (microbial transglutaminase) isolated from the bacterium Streptomyces mobaraensis has been used extensively throughout the food industry to crosslink proteins together for various applications. Besides its low manufacturing cost, it is an attractive conjugation technique due to its ability to function under a wide range of pH, salt, and temperature conditions.

Despite over two decades of research, the substrate specificity of microbial transglutaminase has not been clearly defined. In general, glutamines or lysines on exposed loops with hydrophobic or positively charged adjacent residues tend to be preferred. See, Taguchi et al., “Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates”, J. Biochem. (2000) 128:415-425; Sugimura et al., “Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library”, Arch. Biochem. Biophys. (2008) 477:379-383; Tagami et al., “Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis”, Protein Eng. Des. Sel. (2009) 22:747-752. The context of the acyl donor glutamine has been found to be more critical than the acyl acceptor lysine. See, e.g., Ohtsuka et al., “Substrate specificities of microbial transglutaminase for primary amines”, J. Agric. Food Chem. (2000) 48: 6230-6233; Ohtsuka et al., “Comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors”, Biosci. Biotechnol. Biochem. (2000) 64: 2608-2613; Gundersen et al., “Microbial transglutaminase displays broad acyl-acceptor substrate specificity”, Appl. Microbiol. Biotechnol. (2013) 98:219-230. Indeed, a minimal acyl donor substrate requires an N-terminal N-carboxybenzyloxl (CBZ) group followed by a glutamine and a C-terminal glycine (CBZ-L-glutaminylglycine or Z-Gln-Gly) while the minimal acyl acceptor is ammonia.

Due to a lower specificity for the acyl acceptor amine by microbial transglutaminase, research thus far has been focused mainly on transamidation of antibody glutamine residues. See, Josten et al., Mindt et al., Jeger et al., Strop et al., Dennler et al., and Siegmund et al., referenced above. Contrary to an earlier report (Josten et al. 2000) describing mTGase-mediated biotinylation of an antibody using acyl acceptor substrates, several groups recently showed little or no mTGase modification of wild-type antibodies by the same or similar substrates. These data confirmed that despite the abundance of solvent-exposed glutamines, none were in the proper context to be transamidated by mTGase (Mindt et al. 2008; Jeger et al. 2010; Strop et al. 2013).

It has also been speculated that utilizing an amine donor-based substrate to transamidate a lysine may yield a heterogeneous ADC product due to multiple reactive lysines on the surface of an IgG (Josten et al. 2000; Jeger et al. 2010). Human IgG is comprised of an average of 80 lysines, of which 80-90% are predicted to be solvent exposed (Gautier et al., “Lysine Conjugated Properties in Human IgGs Studied by Integrating High-Resolution Native Mass Spectrometry and Bottom-Up Proteomics”, Proteomics (2015) 15(16):2756-2765; data not shown), and the C-terminal codon of IgG₁, IgG₂, IgG₃, and IgG₄ is a lysine (Ellison et al., DNA (1981) 1:11-18; Ellison et al. (“Ellison et al. 2”), Proc. Nat. Acad. Sci. USA, (1982) 79:1984-1988; Ellison et al., Nucleic Acid Res. (1982) 10:4071-4079). However, serum-derived IgG lacks the lysine (Wang et al., J. Immunol. (1980) 125:1048-1054; Edelman et al., Proc Natl Acad. Sci. USA (1969) 63:78-85; Frangione et al., Biochemistry (1980) 19:4304-4308; Pink et al., Biochem. J. (1970) 117:33-47). The same has been observed for IgD (White et al., Science (1985) 228:733-737; Lin et al., Proc. Natl. Acad. Sci. USA, (1981) 78:504-508; Shinoda et al., Proc. Natl. Acad. Sci. USA (1981) 78:785-789). Recombinant expression of IgG1 in HEK293 and CHO cells also results in a protein lacking the C-terminal Lys447 (Ellison et al.; Harris et al., Eur. J. Biochem. (1990) 194:611-620; Harris, J. Chromatogr. A (1995) 705:129-134; Dick et al., Biotechnol. Bioeng. (2008) 100:1132-1143).

To date, those of ordinary skill in the art thought that utilizing an amine donor-based substrate to transamidate a lysine may yield a heterogeneous ADC product due to the plethora of reactive lysines on the surface of an IgG (Josten et al. and Jeger et al.) and, thus, use of an amine donor-based substrate to transamidate lysine residues on immunoglobulins has been discouraged.

Thus, there exists a need for site-specific enzymatic modifications of immunoglobulins to create conjugates which have a predictable rate of conjugation. This will allow for creation of ADCs with a relatively homologous DAR.

SUMMARY

The instant invention surprisingly discloses that, while no modification of wild-type immunoglobulin lysines by microbial transglutaminase was observed, when an engineered lysine residue was introduced into the immunoglobulin, or antigen-binding portion thereof, microbial transglutaminase was able to utilize the engineered lysine residue as an acyl acceptor. Surprisingly, conjugation of the engineered lysine residue using microbial transglutaminase leads to site-specific and predictable incorporation of conjugated functional agents. Moreover, engineering lysine residues into the constant region has a wide applicability to any immunoglobulin, or antigen-binding portion thereof, regardless of its variable regions and binding specificity.

In one aspect, disclosed herein is a method for generating a conjugated immunoglobulin, the method comprising contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and a functional agent comprising an acyl donor substrate and wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, wherein the acyl donor substrate comprises a glutamine residue, and wherein the functional agent is a therapeutic agent or a diagnostic agent, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate on the functional agent, thereby generating the conjugated immunoglobulin.

In another aspect, disclosed herein is a method for generating a conjugated immunoglobulin, the method comprising i) contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and an acyl donor substrate, wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue and wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, and wherein the acyl donor substrate comprises a glutamine residue and a reactive group, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate, and ii) conjugating a functional agent to the reactive group of the acyl donor substrate, wherein the functional agent is a therapeutic agent or a diagnostic agent, thereby generating the conjugated immunoglobulin. In one embodiment, the engineered lysine residue is present in a heavy chain. In one embodiment, the engineered lysine residue is present in a heavy chain constant region. In one embodiment, the engineered lysine residue is present in a light chain. In another embodiment, the the engineered lysine residue is present in a light chain constant region. In one embodiment, the engineered lysine residue is present in a kappa light chain chain. In one embodiment, the engineered lysine residue is present in a kappa light chain constant region. In one embodiment, the engineered lysine residue is present in a lambda light chain. In one embodiment, the engineered lysine residue is present in a lambda light chain constant region. In one embodiment, the engineered lysine residue is present in a variable region. In one embodiment, the engineered lysine residue is not present in a variable region.

In one embodiment, the reactive group of the acyl donor substrate is conjugated to the functional agent by click chemistry.

In one embodiment, the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Threonine 135 (T135K), Serine 136 (S136K), Leucine 193 (L193K), Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Methionine 252 (M252K), Asparagine 297 (N297K), or Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, Leucine 201 (L201K) or Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, or Glutamic acid 213 (E213K) on a lambda light chain of the immunoglobulin, or antigen-binding portion thereof. In one embodiment, the heavy chain (before mutation) comprises an amino acid sequence set forth as SEQ ID NO:18. In another embodiment, the kappa light chain (before mutation) comprises an amino acid sequence set forth as SEQ ID NO:19. In another embodiment, the lambda light chain (before mutation) comprises a sequence set forth as SEQ ID NO:20.

In one embodiment, the heavy chain further comprises an amino acid residue which has been added to its C-terminus at position 448, and wherein said amino acid residue is not proline or an acidic amino acid residue. In a further embodiment, the amino acid residue which has been added to the C-terminus at position 448 is leucine.

In one embodiment, the lysine residue insertion is a lysine residue which has been inserted between Serine 191 and Serine 192 or between Serine 192 and Leucine 193 on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin is a fragment-antigen binding (Fab′), and wherein the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Proline 228 (P228K), Proline 230 (P230K), and Glutamic acid 233 (E233K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof. In another embodiment, the Fab′ comprises the entire hinge region. In another embodiment, the Fab′ comprises a truncated hinge region.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, further comprises a second engineered lysine residue, wherein the second engineered lysine residue is a second lysine residue insertion or a second natural amino acid residue which has been mutated to a lysine residue, and wherein the microbial transglutaminase conjugates the second engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In another embodiment, the natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof. In another embodiment, the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Leucine 201 (L201K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof. In yet another embodiment, the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof. In a further embodiment, the heavy chain further comprises an amino acid residue which has been added to its C-terminus at position 448, and wherein said amino acid residue is not proline or an acidic amino acid residue. In one further aspect, the amino acid residue which has been added to the C-terminus at position 448 is leucine.

In one embodiment, the immunoglobulin further comprises a third engineered lysine residue, wherein the third engineered lysine residue is a third lysine residue insertion or a third natural amino acid residue which has been mutated to a lysine residue, and wherein the microbial transglutaminase conjugates the third engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In one embodiment, the first natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second engineered lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the third natural amino acid residue which has been mutated to the third engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, further comprises a fourth engineered lysine residue, wherein the fourth engineered lysine residue is a fourth lysine residue insertion or a fourth natural amino acid residue which has been mutated to a lysine residue, and wherein the microbial transglutaminase conjugates the fourth engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In another embodiment, the first natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second engineered lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the third natural amino acid residue which has been mutated to the third engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, and the fourth natural amino acid residue which has been mutated to a fourth engineered lysine residue is Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the amino acid residue after the engineered lysine residue is not proline or an acidic amino acid residue. In another embodiment, the amino acid residue before the engineered lysine residue is not an acidic amino acid residue. In another embodiment, mutating the amino acid residue after the engineered lysine residue to any amino acid other than proline or an acidic amino acid residue and mutating the amino acid residue before the engineered lysine residue to any amino acid other than an acidic amino acid residue, provide the optimal sequence for an engineered acyl acceptor lysine site.

In one embodiment, the amino acid residue before the engineered lysine residue is a non-acidic amino acid residue insertion, or a natural acidic amino acid residue which has been mutated to a non-acidic amino acid residue. In one embodiment, the amino acid residue after the engineered lysine residue is an amino acid residue insertion, wherein the amino acid residue insertion is a non-acidic amino acid residue insertion and a non-proline residue insertion, or a natural acidic amino acid residue or a natural proline residue, which has been mutated to a non-acidic amino acid residue and a non-proline residue. In one embodiment, the non-acidic amino acid residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the non-acidic amino acid residue insertion is an lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan insertion. In one embodiment, the non-acidic amino acid and non-proline residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the natural acidic amino acid residue is aspartic acid or glutamic acid.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a heavy chain which further comprises at least one amino acid residue which has been added to its C-terminus at position 448, and wherein said at least one amino acid residue is not proline or an acidic amino acid residue. In a further embodiment, the at least one amino acid residue which has been added to the C-terminus at position 448 is leucine.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises an insertion of one to four additional amino acids after cysteine 214, wherein the lysine residue insertion is a lysine residue which has been inserted after the one to four additional amino acids, and wherein a leucine residue has been inserted after the lysine residue. In a further embodiment, the insertion of one to four additional amino acids after cysteine 214, the lysine residue which has been inserted after the one to four additional amino acids, and the leucine residue which has been inserted after the lysine residue comprises a sequence selected from the group consisting of: GKL, GGKL (SEQ ID NO: 285), GGSKL (SEQ ID NO: 286), and GGSGKL (SEQ ID NO: 287). In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises an insertion of the sequence GGSGKL (SEQ ID NO: 285) after cysteine 214.

In one embodiment, the functional agent comprising the acyl donor substrate is according to one Formulae (I) or (II):

(Z)_(m)-Gln-(L)_(n)-(Y)  (I)

(Y)-(L)_(n)-Gln-(Z)_(m)  (II)

wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and Y is a functional agent.

In one embodiment, the functional agent comprising the acyl donor substrate is according to formula (I), and wherein Z is a CBZ group; wherein L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2 or 3. In one embodiment, L is a polyethylene glycol moiety (PEG). In another embodiment, L comprises one or more amino acids and a polyethylene glycol moiety (PEG). In another embodiment, the functional agent comprising the acyl donor substrate is according to formula (I), wherein Z is a CBZ group, and wherein L is an amino acid. In one embodiment, L is Gly; m is 1; and n is 1. In another embodiment, the functional agent comprising the acyl donor substrate is according to formula (II), wherein Z is a CBZ group; m is 1; n is 2, 3 or 4; and at least one L is Gly; and at least one L is a PEG moiety. In a further embodiment, the functional agent comprising the acyl donor substrate is according to formula (II), wherein Z is a CBZ group; m is 1; n is 4; one L is Gly and the remaining three L groups are each PEG moieties. In another embodiment, the functional agent Y is auristatin F.

In one embodiment, the acyl donor substrate is according to one Formulae (III) or (IV):

(Z)_(m)-Gln-(L)_(n)-(X)  (III)

(X)-(L)_(n)-Gln-(Z)_(m)  (IV)

wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and X is a reactive group.

In one embodiment, L is a polyethylene glycol moiety (PEG). In another embodiment, when n is 2-5, at least one L comprises one or more amino acids and another L is a polyethylene glycol (PEG) moiety. In one embodiment, the acyl donor substrate is according to formula (III), and wherein Z is a CBZ group; wherein L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2 or 3. In another embodiment, the acyl donor substrate is according to formula (III), wherein Z is a CBZ group, and wherein L is an amino acid. In one embodiment, L is Gly; n is 1; and m is 1. In another embodiment, the acyl donor substrate is according to formula (IV), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is Gly.

In another embodiment, X is a reactive group selected from the group consisting of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN),

(dibenzocyclooctyne; DBCO), trans-cyclooctene (TCO), azido (N₃), alkyne, tetrazine methylcyclopropene, norbornene, hydrazide/hydrazine, and aldehyde.

In one embodiment, the therapeutic agent is an antibody or antigen-binding portion thereof, a chemotherapeutic agent, a drug agent, a radioactive agent, a cytotoxic agent, an antibiotic, a small molecule, a nucleic acid, or a polypeptide. In another embodiment, the diagnostic agent is a fluorophore, a fluorescent dye, a radionuclide, or an enzyme.

In one embodiment, the microbial transglutaminase is from Streptomyces mobaraensis.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof is an IgG₁ immunoglobulin, or antigen-binding portion thereof. In another embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgG₂, IgG₃, or IgG₄ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the immunoglobulin is an IgA₁, an IgA₂, or an IgM immunoglobulin. In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgD or IgE, immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is a human immunoglobulin, or antigen-binding portion thereof or a humanized immunoglobulin, or antigen-binding portion thereof. In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is a chimeric immunoglobulin or a non-human immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises two heavy chains and two light chains. In one embodiment, there is no intramolecular cross-linking, i.e., no disulfide bond(s), between the two heavy chains of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the ratio of functional agent to immunoglobulin, or antigen-binding portion thereof, is 1:1 to 200:1 or 1:1 to 100:1.

In another aspect, described herein is a conjugated immunoglobulin comprising an immunoglobulin, or antigen-binding portion thereof and a functional agent, wherein the immunoglobulin comprises an engineered lysine residue and wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, the functional agent comprises an acyl donor substrate, wherein the acyl donor substrate comprises a glutamine residue, and the functional agent is a therapeutic agent or a diagnostic agent, wherein the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, is conjugated to the glutamine residue of the acyl donor substrate of the functional agent. In one embodiment, the engineered lysine residue is present in a heavy chain. In one embodiment, the engineered lysine residue is present in a heavy chain constant region. In one embodiment, the engineered lysine residue is present in a light chain. In another embodiment, the the engineered lysine residue is present in a light chain constant region. In one embodiment, the engineered lysine residue is present in a kappa light chain chain. In one embodiment, the engineered lysine residue is present in a kappa light chain constant region. In one embodiment, the engineered lysine residue is present in a lambda light chain. In one embodiment, the engineered lysine residue is present in a lambda light chain constant region. In one embodiment, the engineered lysine residue is present in a variable region. In one embodiment, the engineered lysine residue is not present in a variable region.

In another aspect, described herein is a conjugated immunoglobulin comprising an immunoglobulin, or antigen-binding portion thereof and a functional agent, wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue and wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, the engineered lysine residue is conjugated to a glutamine residue on an acyl donor substrate, wherein the acyl donor substrate further comprises a reactive group, the reactive group is conjugated to a functional agent, wherein the functional agent is a therapeutic agent or a diagnostic agent. In one embodiment, the engineered lysine residue is present in a heavy chain. In one embodiment, the engineered lysine residue is present in a heavy chain constant region. In one embodiment, the engineered lysine residue is present in a light chain. In another embodiment, the the engineered lysine residue is present in a light chain constant region. In one embodiment, the engineered lysine residue is present in a kappa light chain chain. In one embodiment, the engineered lysine residue is present in a kappa light chain constant region. In one embodiment, the engineered lysine residue is present in a lambda light chain. In one embodiment, the engineered lysine residue is present in a lambda light chain constant region. In one embodiment, the engineered lysine residue is present in a variable region. In one embodiment, the engineered lysine residue is not present in a variable region.

In one embodiment, the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Threonine 135 (T135K), Serine 136 (S136K), Leucine 193 (L193K), Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Methionine 252 (M252K), Asparagine 297 (N297K), or Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, Leucine 201 (L201K) or Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, or Glutamic acid 213 (E213K) on a lambda light chain of the immunoglobulin, or antigen-binding portion thereof. In one embodiment, the heavy chain (before mutation) comprises an amino acid sequence of SEQ ID NO:18. In one embodiment, the kappa light chain (before mutation) comprises an amino acid sequence of SEQ ID NO:19. In one embodiment, the lambda light chain (before mutation) comprises an amino acid sequence of SEQ ID NO:20.

In one embodiment, the heavy chain further comprises an amino acid residue which has been added to its C-terminus at position 448, and wherein said amino acid residue is not proline or an acidic amino acid residue. In a further embodiment, the at least one amino acid residue which has been added to the C-terminus at position 448 is leucine.

In another embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises at least one amino acid residue that has been added to the C-terminus after position 214, wherein the at least one amino acid residue is GGSGKL (glycine glycine serine glycine lysine leucine) (SEQ ID NO: 287). In another embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which further comprises at least one amino acid residue which has been added to the C-terminus after position 214, wherein the at least one amino acid residue is GGSGKL (SEQ ID NO: 287). In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises an insertion of the sequence GGSGKL (SEQ ID NO: 287) after cysteine 214.

In one embodiment, the lysine residue insertion is a lysine residue which has been inserted between Serine 191 and Serine 192 or between Serine 192 and Leucine 193 on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof is a fragment-antigen binding (Fab), and wherein the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Proline 228 (P228K), Proline 230 (P230K), and Glutamic acid 233 (E233K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof. In another embodiment, the Fab comprises the entire hinge region. In another embodiment, the Fab comprises a truncated hinge region.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, further comprises a second natural amino acid residue which has been mutated to a second lysine residue, and wherein the microbial transglutaminase conjugates the second lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In another embodiment, the first natural amino acid residue which has been mutated to the lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second lysine residue is Serine 202 (S202K). In another embodiment, the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Leucine 201 (L201K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof. In yet another embodiment, the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.

In a further embodiment, the heavy chain further comprises at least one amino acid residue which has been added to its C-terminus at position 448, and wherein the at least one amino acid residue is not proline or an acidic amino acid residue. In one embodiment, the at least one amino acid residue which has been added to the C-terminus at position 448 is leucine.

In one embodiment, the immunoglobulin further comprises a third natural amino acid residue which has been mutated to a third lysine residue, and wherein the microbial transglutaminase conjugates the third lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In another embodiment, the first natural amino acid residue which has been mutated to the lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the third natural amino acid residue which has been mutated to the third lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, further comprises a fourth natural amino acid residue which has been mutated to a fourth lysine residue, and wherein the microbial transglutaminase conjugates the fourth lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate. In another embodiment, the first natural amino acid residue which has been mutated to the lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the third natural amino acid residue which has been mutated to the third lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, and the fourth natural amino acid residue which has been mutated to a fourth lysine residue is Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the amino acid residue after the engineered lysine residue is not proline or an acidic amino acid residue. In another embodiment, the amino acid residue before the engineered lysine residue is not an acidic amino acid residue. In another embodiment, mutating the amino acid residue after the engineered lysine residue to any amino acid other than proline or an acidic amino acid residue and mutating the amino acid residue before the engineered lysine residue to any amino acid other than an acidic amino acid residue, provide the optimal sequence for an engineered acyl acceptor lysine site.

In one embodiment, the amino acid residue before the engineered lysine residue is a non-acidic amino acid residue insertion, or a natural acidic amino acid residue which has been mutated to a non-acidic amino acid residue. In one embodiment, the amino acid residue after the engineered lysine residue is an amino acid residue insertion, wherein the amino acid residue insertion is a non-acidic amino acid residue and a non-proline residue insertion, or a natural acidic amino acid residue or a natural proline residue, which has been mutated to a non-acidic amino acid residue and a non-proline residue. In a further embodiment, the non-acidic amino acid residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In another further embodiment, the non-acidic amino acid residue insertion is an lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan insertion. In yet another further embodiment, the non-acidic amino acid and non-proline residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In yet another further embodiment, the natural acidic amino acid residue is aspartic acid or glutamic acid.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a heavy chain which further comprises at least one amino acid residue which has been added to its C-terminus at position 448, and wherein the at least one amino acid residue is not proline or an acidic amino acid residue. In a further embodiment, the at least one amino acid residue which has been added to the C-terminus at position 448 is leucine.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises an insertion of one to four additional amino acids after cysteine 214, wherein the lysine residue insertion is a lysine residue which has been inserted after the one to four additional amino acids, and wherein a leucine residue has been inserted after the lysine residue. In a further embodiment, the insertion of one to four additional amino acids after cysteine 214, the lysine residue which has been inserted after the one to four additional amino acids, and the leucine residue which has been inserted after the lysine residue comprise a sequence selected from the group consisting of: GKL, GGKL (SEQ ID NO: 285), GGSKL (SEQ ID NO: 286), and GGSGKL (SEQ ID NO: 287).

In one embodiment, the functional agent comprising the acyl donor substrate is according to one Formulae (I) or (II):

(Z)_(m)-Gln-(L)_(n)-(Y)  (I)

(Y)-(L)_(n)-Gln-(Z)_(m)  (II)

wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and Y is a functional agent.

In one embodiment, the functional agent comprising the acyl donor substrate is according to formula (I), and wherein Z is a CBZ group; wherein L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2 or 3. In another embodiment, the functional agent comprising the acyl donor substrate is according to formula (I), wherein Z is a CBZ group, and wherein L is an amino acid. In one embodiment, L is Gly; m is 1; and n is 1. In one embodiment, the functional agent comprising the acyl donor substrate is according to formula (II), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is Gly. In one embodiment, L is a polyethylene glycol moiety (PEG). In another embodiment, L comprises one or more amino acids and a polyethylene glycol moiety (PEG). In another embodiment, the functional agent Y is auristatin F.

In one embodiment, the acyl donor substrate is according to one Formulae (III) or (IV):

(Z)_(m)-Gln-(L)_(n)-(X)  (III)

(X)-(L)_(n)-Gln-(Z)_(m)  (IV)

wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and X is a reactive group.

In one embodiment, the acyl donor substrate is according to formula (III), and wherein Z is a CBZ group; wherein L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2 or 3. In another embodiment, the acyl donor substrate is according to formula (III), wherein Z is a CBZ group, and wherein L is an amino acid. In one embodiment, L is Gly; m is 1; and n is 1. In another embodiment, the acyl donor substrate is according to formula (IV), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is Gly. In one embodiment, L is a polyethylene glycol moiety (PEG). In another embodiment, when n is 2-5, then at least one L comprises one or more amino acids and one or more L comprises a polyethylene glycol moiety (PEG).

In one embodiment, X is a reactive group selected from the group consisting of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN),

(dibenzocyclooctyne; DBCO), trans-cyclooctene (TCO), azido (N₃), alkyne, tetrazine methylcyclopropene, norbornene, hydrazide/hydrazine, and aldehyde.

In one embodiment, the therapeutic agent is an antibody or antigen-binding portion thereof, a chemotherapeutic agent, a drug agent, a radioactive agent, a cytotoxic agent, an antibiotic, a small molecule, nucleic acid, or a polypeptide. In another embodiment, the diagnostic agent is a fluorophore, a fluorescent dye, a radionuclide, or an enzyme.

In one embodiment, the microbial transglutaminase is from Streptomyces mobaraensis.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgG₁ immunoglobulin, or antigen-binding portion thereof. In another embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgG₂, IgG₃, or IgG₄ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgA₁, an IgA₂, or an IgM immunoglobulin, or antigen-binding portion thereof. In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is an IgD or IgE, immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof is a human immunoglobulin, or antigen-binding portion thereof, or a humanized immunoglobulin, or antigen-binding portion thereof. In one embodiment, the immunoglobulin, or antigen-binding portion thereof, is a chimeric immunoglobulin or a non-human immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the immunoglobulin, or antigen-binding portion thereof, comprises two heavy chain and two light chains. In one embodiment, there is no intramolecular cross-linking between the two heavy chains of the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the ratio of functional agent to immunoglobulin, or antigen-binding portion thereof is 1:1 to 200:1 or 1:1 to 100:1.

In one embodiment, the functional agent is an antibody, or antigen-binding portion thereof, and wherein the immunoglobulin, or antigen-binding portion thereof, and the functional agent bind the same antigen or bind different antigens.

In another aspect, described herein is a nucleic acid encoding a conjugatable immunoglobulin. In another aspect, described herein is a plasmid comprising a nucleic acid. In another embodiment, described herein is an isolated cell comprising a plasmid.

In another aspect, described herein is a pharmaceutical composition comprising a conjugated immunoglobulin and a pharmaceutically acceptable carrier.

In one aspect, described herein is a conjugated immunoglobulin produced by any of the methods described herein. In one embodiment, the method further comprises a step of purifying the immunoglobulin conjugated to the glutamine residue of the acyl donor substrate before conjugating the functional agent to the reactive group of the acyl donor substrate. In one embodiment, the purifying step comprises size-based methods, such as chromatography or diafiltration. In another embodiment, the purifying step includes charge-based separation, such as anion exchange or cation exchange chromatography. In another embodiment, the purifying step comprises an affinity-based step, such as Protein A or Protein G chromatography and hydrophobic interaction chromatography (HIC).

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, and conjugated immunoglobulins, there are shown in the drawings exemplary embodiments; however, the methods and conjugated immunoglobulins are not limited to the specific embodiments disclosed. In the drawings:

FIG. 1 shows a transglutaminase reaction, wherein the transglutaminase catalyzes the formation of an isopeptide bond between an acyl donor glutamine and an acyl acceptor lysine with release of an ammonia molecule.

FIG. 2 shows the structures of exemplary Z-Gln-Gly acyl-donor substrates.

FIG. 3 shows possible routes to synthesize exemplary Z-Gln-Gly acyl-donor substrates.

FIG. 4, Panels A, B and C, show solvent exposed lysines (stick representation) in human IgG₁ Fab and Fc crystal structures; (Panel A) Fab VH-CH1 and Vκ-Cκ, (Panel B) Fab VH-CH1 and Vλ-Cλ, and (Panel C) Fc CH2 and CH3 were determined using Discovery Studio 4.5 with a 1.4 Å probe radius.

FIG. 5, Panels A, B, C, D, E, and F, show ESI-MS analysis of antibodies contacted with an acyl donor and microbial transglutaminase. Antibodies were contacted with 50-fold molar excess Z-Gln-Gly-CAD-biotin and 1 U/mL microbial transglutaminase overnight at 37° C. Following IdeS digestion and reduction, the LC, Fd, and Fc masses were determined by ESI-MS.

FIG. 6 (SEQ ID NOS: 288-290) shows sequences of human IgG₁, kappa, and lambda constant domains. Solvent exposed constant domain lysines based on the crystal structures 1FC1 (Fcγ), 4F3F (CH1 and Cκ), and 4HK0 (Cλ) are in bold; lysines within loops are underlined. The constant domains are numbered according the EU numbering system.

FIG. 7 shows the conjugation levels of purified CH1 and hinge mutant mAbs screened for transamidation by incubating mTGase with Z-Gln-Gly-CAD-biotin overnight at 37° C. Biotinylated mAbs were detected by an ELISA as detailed in the Material and Methods and the relative fluorescent units (RFUs) were analyzed. The masses of the HC and LC were analyzed by ESI-MS (data not shown) and the percentage of conjugation was determined.

FIG. 8 shows the conjugation levels of CH1, CH2, CH3 and hinge mutant mAbs previously identified as having the highest conjugation efficiency. MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight. The samples were digested with IdeS, reduced, and analyzed by ESI-MS, and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass=631 Da) was determined. The DAR was determined by dividing the Δmass by the mass of Z-Gln-Gly-CAD-biotin

FIG. 9 shows the conjugation effeiciency of mAbs with multiple acyl acceptor cytes with various acyl donors. MAbs were incubated with Z-Gln-Gly-N3, Z-Gln-Gly-PEG2-BCN, or Z-Gln-Gly-PEG2-AuF and mTGase at 37° C. overnight. The masses of the reduced LCs were analyzed by LC-MS (data not shown), and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass=631 Da) was determined.

FIG. 10 (SEQ ID NOS: 291-294) shows the alignment of IgG Fc isotypes. The primary amino acid sequences of human IgG1, 2, 3, and 4 Fc are aligned and differences are highlighted. IgG1 residues Met252, Asn297, and Pro445 are indicated by an X.

FIG. 11 shows Fab's with an acyl acceptor were conjugated to the Q295 and/or Q297 acyl donor site in the mAb mutant. Antibody 01-N297Q was incubated with Fab′ lysine mutants and mTGase overnight at 37° C. The samples were reduced and analyzed by SDS-PAGE using a 4-12% Bis-Tris polyacrylamide gel. The mass of the FabCH1-HC dimer is approximately 75 kDa and the FabCH1-HC-FabCH1 trimer is approximately 100 kDa.

FIG. 12 illustrates engineered acyl acceptor sites located through the mAb. The locations of residues engineered with acyl acceptor cites were highlighted in the (A) Fab (4F3F) HC and LC and (B) Fc (1FC1).

DETAILED DESCRIPTION

The disclosed methods and conjugated immunoglobulins may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods and conjugated immunoglobulins are not limited to the specific embodiments described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods or conjugated immunoglobulins.

Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods and conjugated immunoglobulins are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

Throughout this text, the descriptions refer to conjugated immunoglobulins and methods of generating the same. Where the disclosure describes or claims a feature or embodiment associated with a conjugated immunoglobulin, such a feature or embodiment is equally applicable to the methods of generating the same. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of generating a conjugated immunoglobulin, such a feature or embodiment is equally applicable to the conjugated immunoglobulin.

Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Further, reference to values stated in ranges include each and every value within that range. All ranges are inclusive and combinable.

When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

It is to be appreciated that certain features of the disclosed methods and conjugated immunoglobulins which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods and conjugated immunoglobulins that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

As used herein, the singular forms “a,” “an,” and “the” include the plural.

Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ±10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value.

In general, the term “engineered” refers to the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or by other methods commonly used in the art).

As used herein, the term “engineered amino acid residue” refers to a non-naturally-occurring amino acid residue within the context of a sequence. As used herein, the term “engineered nucleic acid residue” refers to a non-naturally occurring nucleic acid residue within the context of a sequence. These terms include, for example, a polypeptide sequence or nucleic acid sequence that comprises one or more amino acid or nucleotide changes, including additions, deletions or substitutions, relative to the corresponding naturally occurring polypeptide sequence or nucleic acid sequence, wherein such changes were introduced by recombinant DNA techniques. For instance, an “engineered lysine residue” is a lysine residue that did not exist in the corresponding naturally occurring, or wild-type, polypeptide sequence and was introduced into the polypeptide, either by mutating an existing amino acid residue, or by the insertion of a lysine residue, when sequences of the wild type and the engineered version are aligned. An engineered amino acid residue is not a synthetic amino acid residue. For instance, an engineered lysine residue is not a synthetic lysine residue.

In one embodiment, an “engineered amino acid residue” is an amino acid residue insertion. For example, an amino acid residue or residues may be inserted in between two naturally occurring amino acid residues. In another embodiment, an “engineered amino acid residue” is a naturally occurring amino acid residue which has been mutated to a different amino acid residue. For example, an engineered lysine residue may be a naturally-occurring amino acid residue (e.g., histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, or tyrosine) which has been mutated, e.g., using recombination DNA techniques, to a lysine residue. Those skilled in the art can readily generate engineered polypeptide sequence useful according to this aspect of the invention. Engineered polypeptide sequences may be produced by any means, including, for example, peptide, polypeptide, or protein synthesis.

As used herein, the term “insertion” or “addition” refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule (e.g., the wild-type sequence). The insertion or addition of one or more amino acid residues can take place in-between internal amino acid residues. Alternatively, an insertion may occur at the N-terminus of the amino acid sequence. Alternatively, an insertion may occur at the C-terminus of the amino acid sequence.

The term “natural amino acid,” “natural amino acid residue,” “naturally occurring amino acid” or “naturally occurring amino acid residue” refers to naturally occurring amino acids which typically occur within the context of a wild-type polypeptide sequence. In other words, a “natural amino acid” has not been mutated or changed in any way to differ from the amino acid residue present in the parent sequence, e.g., naturally occurring sequence. A “natural amino acid” includes amino acids at any position within the polypeptide sequence (e.g., internal amino acid residues) and also any amino acid at the N-terminus or the C-terminus of the polypeptide sequence.

The term “acyl donor substrate” refers to a group with a terminal acyl group on it. Preferably, the “acyl donor substrate” comprises a glutamine residue. An acyl donor substrate may optionally contain a further reactive group. In a first embodiment, the acyl donor substrate is covalently connected to a functional agent. In a second embodiment, the acyl donor substrate is not connected to a functional agent. In one embodiment, the acyl donor substrate comprises a glutamine residue and a reactive group. In another embodiment, the acyl donor substrate comprises one or more linkers, as described further herein. In any of the above embodiments, there is optionally a linker between the acyl donor substrate and the functional agent or between the acyl donor substrate and the reactive group.

The term “antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains. The term “antibody”, as used herein, also refers to any antigen-binding portion, mutant, variant, or derivative of an immunoglobulin molecule, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art and nonlimiting embodiments of which are discussed herein. In one embodiment, the antibody is a humanized antibody. In another embodiment, the antibody is a human antibody. In another embodiment, the antibody is a chimeric antibody. In another embodiment, the antibody is a non-human antibody.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHL CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass.

The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a Fab′ fragment, a Fab containing hinge region not linked by disulfide via either mild reduction or by mutating or deleting cysteines; (iii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

“Acidic Amino Acid” refers to an amino acid exhibiting a negative charge at physiological pH. Genetically encoded acidic amino acids include aspartic acid (Asp; D) and glutamic acid (Glu; E).

“Non-Acidic Amino Acid” refers to an amino acid which is not an acidic amino acid. Non-acidic amino acids include arginine (Arg; R), histidine (His; H), lysine (Lys; K), serine (Ser; S); threonine (Thr; T), asparagine (Asn; N), glutamine (Gln; Q), cysteine (Cys; C), glycine (Gly; G), proline (Pro; P), alanine (Ala; A), valine (Val; V); isoleucine (Ile; I), leucine (Leu; L), methionine (Met; M), phenylalanine (Phe; F), tyrosine (Tyr; Y), and tryptophan (Trp; W).

“Basic Amino Acid” refers to an amino acid exhibiting a positive charge at physiological pH. Genetically encoded basic amino acids include histidine (His; H), lysine (Lys, K), and arginine (Arg; R).

As used herein, the term “biological sample” refers to a sample obtained from a subject, including sample of biological tissue or fluid origin obtained in vivo or in vitro. Such samples can be, but are not limited to, body fluid (e.g., blood, blood plasma, serum, milk, spinal fluid, ascites, or urine), organs, tissues, fractions, and cells isolated from mammals including, humans Biological samples also may include sections of the biological sample including tissues (e.g., sectional portions of an organ or tissue). Biological samples may also include extracts from a biological sample, for example, an antigen from a biological fluid (e.g., blood or urine).

The term “click chemistry” refers to particular reactions for protein synthesis and/or conjugation which are high yield, highly-selective, reliable and clean. See, e.g., King et al., “Developments in the Field of Bioorthagonal Bond Forming Reactions—Past and Present Trends”, Bioconjug. Chem., (2014) 25(5): 825-839; McKay et al., “Click Chemistry in Complex Mixtures: Bioorthagonal Bioconjugation”, Chem. Biol., (2014) 21(9): 1075-1101.

The term “chimerized,” “chimeric,” “chimeric antibody” and like terms refer to an immunoglobulin comprising a heavy chain variable region and light chain variable region, i.e., antigen-binding region, from one source or species and at least a portion of a heavy chain constant region and light chain constant region derived from a different source or species. These portions may be joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody may be expressed to produce a contiguous polypeptide chain) Other forms of “chimeric immunoglobulins” encompassed by the present disclosure are those in which the class or subclass has been modified or changed from that of the original immunoglobulin (also referred to as “class-switched immunoglobulins”). Throughout the disclosure, chimeric immunoglobulins are designated “xi.” Herein, “chimeric immunoglobulin” and like terms refer to the sequence of the immunoglobulin rather than the process used to generate the antibody.

As used herein, “functional agent” refers to an agent having therapeutic, diagnostic, or other functional property(ies). In one embodiment, a functional agent may be a therapeutic agent. In another embodiment, a functional agent may be a diagnostic agent. Functional agents may be large molecules or small molecules. Large molecule functional agents include, but are not limited to, an antibody and antigen-binding portions thereof. Small molecule functional agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, antibiotics, other organic compounds which may regulate biological process (e.g., drugs), and polypeptides.

The term “humanized,” “humanized immunoglobulin” and like terms refer to immunoglobulins in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. For the most part, humanized immunoglobulins are human immunoglobulins (recipient immunoglobulin) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor immunoglobulin) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized immunoglobulins may comprise residues that are not found in the recipient immunoglobulin or in the donor immunoglobulin. These modifications are made to further refine immunoglobulin performance. In general, the humanized immunoglobulin will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized immunoglobulin can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270. Herein, “humanized immunoglobulin” and like terms refer to the sequence of the immunoglobulin rather than the process used to generate the immunoglobulin.

The term “diagnostic agent” refers to a compound which may be useful for in vivo imaging studies such as CT, MRI and X-ray and/or in vitro imaging studies. Non-limiting examples of diagnostic agents include a fluorophore, a fluorescent dye, a radionuclide, and an enzyme.

The term “donor immunoglobulin” refers to a non-human immunoglobulin that contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to the humanized immunoglobulin, and thereby provides the humanized immunoglobulin with the antigenic specificity and neutralizing activity characteristic of the donor immunoglobulin.

The term “recipient immunoglobulin” refers to an immunoglobulin heterologous to the donor immunoglobulin, which provides the amino acid sequences of its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the humanized immunoglobulin. The recipient immunoglobulin may be derived from any mammal. In preferred embodiments, the recipient immunoglobulin is non-immunogenic in humans Preferably the recipient immunoglobulin is a human immunoglobulin.

“Humanizing” refers to a process of generating a humanized immunoglobulin and includes any process for generating humanized immunoglobulins having the above characteristics, including, but not limited to, in silico humanization, engineering species/host CDRs into human immunoglobulins, substituting framework region residues of a chimeric immunoglobulin to match a corresponding human framework region, etc.

“Immunoglobulin,” as used herein, refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes including the kappa and lambda light chains and the alpha, gamma, delta, epsilon and mu heavy chains. Full-length immunoglobulin “light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH₂-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). “Immunoglobulins” include: (a) immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family that contain an antigen binding site that specifically binds to a specific antigen, including all immunoglobulin isotypes (IgG, IgA, IgE, IgM, IgD, and IgY), classes (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂), subclasses, and various monomeric and polymeric forms of each isotype, unless otherwise specified; and (b) conservatively substituted variants of such immunoglobulin polypeptides that immunospecifically bind to the antigen Immunoglobulins are generally described in, for example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

The term “microbial transglutaminase” refers to a type of transferase that catalyzes an acyl transfer reaction. A preferred embodiment comprises the use of a microbial transglutaminase to catalyze an acyl transfer reaction between a first moiety containing a glutamine residue (acyl donor) and a second moiety containing a primary amine group (acyl acceptor). It is preferable that the reactive glutamine residue is solvent exposed.

One form of immunoglobulin disclosed herein constitutes the basic structural unit of an antibody. For example, an antibody can include a tetramer and consist of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. Generally, in each pair, the light chain and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.

In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example: antigen-binding fragments or portions of a full-length immunoglobulin, such as Fv, Fab, (Fab′)₂ and Fv fragments; and alternative antibody formats such as single chain immunoglobulins (scFV and scFab), diabodies, triabodies, tetrabodies, linear antibodies, and multispecific antibodies, to name a few. See, for example, James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).

In one embodiment, an immunoglobulin may comprise an Fab fragment. In another embodiment, an immunoglobulin may comprise a CH3 domain. In another embodiment, an immunoglobulin may comprise a heavy chain.

As used herein, the term “immunospecifically” refers to the ability of an immunoglobulin to specifically bind to an antigen against which the immunoglobulin was generated and not specifically bind to other peptides or proteins. An immunoglobulin that immunospecifically binds to an antigen against which the immunoglobulin was generated may not bind to other polypeptides or proteins, or may bind to other polypeptides or proteins with a lower binding affinity than the antigen against which the immunoglobulin was generated as determined by, for example, immunoassays, BIAcore, or other assays known in the art. An immunoglobulin binds immunospecifically to an antigen against which the immunoglobulin was generated when it binds to the antigen with a higher binding affinity than to any cross-reactive antigen as determined using experimental techniques, such as, but not limited to, radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs) (See, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989) for a discussion regarding antibody specificity.).

“Linker,” as used herein, refers to a spacer, which may be a straight or branched chain, for connecting an immunoglobulin (through an acyl donor substrate) to a functional agent or a reactive group. Such linkers may be cleavable (e.g., acid labile or protease cleavable) or non-cleavable. In one embodiment, a linker is a polyethylene glycol (PEG) moiety. In another embodiment, a linker comprises one or more amino acids and a polyethylene glycol moiety (PEG).

The term “monoclonal antibody” refers to an antibody that is derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or a phage clone, and not the method by which it is produced. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.

“Native” refers to the wild type immunoglobulin sequence from the species in which the immunoglobulin is derived.

As used herein, “percent identity” and like terms is used to describe the sequence relationships between two or more nucleic acids, polynucleotides, proteins, or polypeptides, and is understood in the context of and in conjunction with the terms including: (a) reference sequence, (b) comparison window, (c) sequence identity and (d) percentage of sequence identity.

-   -   (a) A “reference sequence” is a defined sequence used as a basis         for sequence comparison. A reference sequence may be a subset of         or the entirety of a specified sequence; for example, a segment         of a full-length cDNA or gene sequence, or the complete cDNA or         gene sequence. For polypeptides, exemplary lengths of the         reference polypeptide sequence include at least about 16 amino         acids, at least about 20 amino acids, at least about 25 amino         acids, at least about 35 amino acids, at least about 50 amino         acids, or at least about 100 amino acids. For nucleic acids,         exemplary length of the reference nucleic acid sequence include         at least about 50 nucleotides, at least about 60 nucleotides, at         least about 75 nucleotides, at least about 100 nucleotides, or         at least about 300 nucleotides, or any integer thereabout or         therebetween.     -   (b) A “comparison window” includes reference to a contiguous and         specified segment of a polynucleotide or polypeptide sequence,         wherein the polynucleotide or polypeptide sequence may be         compared to a reference sequence and wherein the portion of the         polynucleotide or polypeptide sequence in the comparison window         may comprise additions, substitutions, or deletions (i.e., gaps)         compared to the reference sequence (which does not comprise         additions, substitutions, or deletions) for optimal alignment of         the two sequences. Exemplary comparison windows can be at least         20 contiguous nucleotides or amino acids in length, and         optionally may be 30, 40, 50, 100, or longer. Those of skill in         the art understand that to avoid a misleadingly high similarity         to a reference sequence due to inclusion of gaps in the         polynucleotide or polypeptide sequence a gap penalty is         typically introduced and is subtracted from the number of         matches.     -   (c) Methods of alignment of sequences for comparison are well         known in the art. Optimal alignment of sequences for comparison         may be conducted by the local homology algorithm of Smith and         Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology         alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:         443, 1970; by the search for similarity method of Pearson and         Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by         computerized implementations of these algorithms, including, but         not limited to: CLUSTAL in the PC/Gene program by         Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST,         FASTA, and TFASTA in the Wisconsin Genetics Software Package,         Genetics Computer Group (GCG), 7 Science Dr., Madison, Wis.,         USA; the CLUSTAL program is well described by Higgins and Sharp,         Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research,         16:881-90, 1988; Huang, et al., Computer Applications in the         Biosciences, 8:1-6, 1992; and Pearson, et al., Methods in         Molecular Biology, 24:7-331, 1994. The BLAST family of programs         which may be used for database similarity searches includes:         BLASTN for nucleotide query sequences against nucleotide         database sequences; BLASTX for nucleotide query sequences         against protein database sequences; BLASTP for protein query         sequences against protein database sequences; TBLASTN for         protein query sequences against nucleotide database sequences;         and TBLASTX for nucleotide query sequences against nucleotide         database sequences. See, Current Protocols in Molecular Biology,         Chapter 19, Ausubel, et al., Eds., Greene Publishing and         Wiley-Interscience, New York, 1995. New versions of the above         programs or new programs altogether will undoubtedly become         available in the future, and may be used with the present         disclosure.     -   (d) “Percent identity” means the value determined by comparing         two optimally aligned sequences over a comparison window,         wherein the portion of the polynucleotide or polypeptide         sequence in the comparison window may comprise additions,         substitutions, or deletions (i.e., gaps) as compared to the         reference sequence (which does not comprise additions,         substitutions, or deletions) for optimal alignment of the two         sequences. The percentage is calculated by determining the         number of positions at which the identical nucleic acid base or         amino acid residue occurs in both sequences to yield the number         of matched positions, dividing the number of matched positions         by the total number of positions in the window of comparison and         multiplying the result by 100 to yield the percentage of         sequence identity.

“Pharmaceutically effective amount” refers to an amount of an immunoglobulin that treats a subject.

“Pharmaceutically acceptable carrier” refers to components of a pharmaceutical formulation for an immunoglobulin as described herein for administration to a subject. For example, a pharmaceutically acceptable carrier may be a liposome-based, lipid-based and/or nano-particle-based.

The term “reactive group” as used here in refers to a chemical functional group which may react to other compounds, such as functional agents, to form at least one covalent bond. In one embodiment, reactive groups are reactive in click chemistry coupling reactions. Non-limiting examples of reactive groups include (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN),

(dibenzocyclooctyne; DBCO), trans-cyclooctene (TCO), azido (N₃), alkyne, tetrazine methylcyclopropene, norbornene, hydrazide/hydrazine, and aldehyde.

The term “subject” as used herein refers to a human or non-human organism. Thus, the methods, immunoglobulins, and conjugated immunoglobulins described herein are applicable to both human and veterinary diseases and conditions. Subjects can be “patients,” i.e., living humans or non-human organisms that are receiving medical care for a disease or condition, or humans or non-human organisms with no defined illness who are being investigated for signs of pathology or presence/absence of a particular condition.

“Substituting” refers to the replacement of one amino acid residue for another. “Substituting” includes, for example, missense mutations in one or more DNA base pairs encoding the amino acid residue or engineering the protein to exchange one amino acid with another.

As used herein, “treating” and like terms refer to reducing the severity and/or frequency of disease symptoms, eliminating disease symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by disease.

The term “therapeutic agent” means a large or small molecule which may be administered to a subject in need thereof to treat a condition. Therapeutic agents may be administered to treat, or prevent the onset, slow the progression, or to ameliorate one or more symptoms of a medical condition in subjects suffering from the same. Therapeutic agents include, but are not limited to, an antibody or antigen-binding portion thereof, a chemotherapeutic agent, a radioactive agent, a cytotoxic agent, an antibiotic, etc. In one embodiment, the therapeutic agent is a small molecule. In another embodiment, the therapeutic agent is a polypeptide.

As used herein “90% identical to” encompasses at least 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to the reference item (e.g., a biological sequence).

The following abbreviations are used throughout the disclosure: antibody drug conjugates (ADCs); drug-to-antibody ratio (DAR); frame work region (FR); complementary determining region (CDR); auristatin F (AuF); variable heavy region (VH); variable light region (VL); variable kappa (Vκ); gamma constant region (Cγ); kappa constant region (CIO; monoclonal antibody (mAb); lysine at amino acid position 447 of the heavy chain of the immunoglobulin, as numbered using the EU numbering system (Lys447).

Generation of Conjugated Immunoglobulins

Disclosed herein are methods for generating a conjugated immunoglobulin, the methods comprising: contacting an immunoglobulin, or an antigen-binding portion thereof, with a microbial transglutaminase and a functional agent comprising an acyl donor substrate, a) wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, b) wherein the acyl donor substrate comprises a glutamine residue, and c) wherein the functional agent is a therapeutic agent or a diagnostic agent, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate on the functional agent, thereby generating the conjugated immunoglobulin.

Also disclosed herein are methods for generating a conjugated immunoglobulin, the methods comprising: i) contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and an acyl donor substrate, a) wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, b) wherein the acyl donor substrate comprises a glutamine residue and a reactive group, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate, and ii) conjugating a functional agent to the reactive group of the acyl donor substrate, wherein the functional agent is a therapeutic agent or a diagnostic agent, thereby generating the conjugated immunoglobulin.

Conjugation can be performed by dissolving a functional agent comprising an acyl donor substrate in a dissolution solution and contacting the dissolved functional agent with the immunoglobulin, or antigen-binding portion thereof, and microbial transglutaminase in a conjugation buffer. Conjugation may also be performed by dissolving a acyl donor substrate in a dissolution solution and contacting the acyl donor substrate with the immunoglobulin, or antigen-binding portion thereof, and microbial transglutaminase in a conjugation buffer.

For aqueous-insoluble functional agents and acyl donor substrates, suitable dissolution solutions include organic, water-miscible solvents such as dimethylsulfoxide (DMSO). For aqueous-soluble functional agents and acyl donor substrates, suitable dissolution solutions include, but are not limited to, water or buffered aqueous solutions, such as phosphate-buffered saline, pH 7.2 (1×PBS) or DPBS.

Suitable concentrations of the functional agent or the acyl donor substrate include from about 10 μM to about 800 mM, from about 10 mM to about 100 mM, from about 25 mM to about 100 mM, from about 40 mM to about 100 mM, from about 55 mM to about 100 mM, from about 70 mM to about 100 mM, from about 10 mM to about 90 mM, from about 10 mM to about 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, or from about 10 mM to about 30 mM.

In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 10 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 25 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 50 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 100 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 250 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 500 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 750 μM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 1 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 10 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 20 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 30 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 40 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 50 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 60 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 70 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 80 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 90 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 100 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 150 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 200 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 250 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 300 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 350 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 400 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 450 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 500 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 550 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 600 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 650 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 700 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 750 mM. In some embodiments, the concentration of the functional agent or the acyl donor substrate can be about 800 mM.

Suitable concentrations of immunoglobulin include from about 0.1 mg/ml to about 100 mg/ml, from about 0.5 mg/ml to about 20 mg/ml, from about 1 mg/ml to about 20 mg/ml, from about 5 mg/ml to about 20 mg/ml, from about 10 mg/ml to about 20 mg/ml, from about 0.1 mg/ml to about 15 mg/ml, from about 0.1 mg/ml to about 12 mg/ml, from about 0.1 mg/ml to about 10 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, or from about 0.1 mg/ml to about 2 mg/ml, from about 10 mg/ml to about 30 mg/ml, from about 20 mg/ml to about 45 mg/ml, from about 35 mg/ml to about 50 mg/ml, from about 45 mg/ml to about 60 mg/ml, from about 50 mg/ml to about 75 mg/ml, from about 60 mg/ml to about 85 mg/ml or from about 80 mg/ml to about 100 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 0.1 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 0.5 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 1 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 2 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 5 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 10 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 15 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 20 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 25 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 30 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 35 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 40 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 45 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 50 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 55 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 60 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 65 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 70 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 75 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 80 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 85 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 90 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 95 mg/ml. In some embodiments, the concentration of immunoglobulin can be about 100 mg/ml.

Suitable ratios of a functional agent or an acyl donor substrate:immunoglobulin include from about 1:1 to 100:1. In one embodiment, the ratio of functional agent to acyl donor substrate:immunoglobulin is about 25:1 to about 75:1. In another embodiment, the ratio of functional agent to acyl donor substrate:immunoglobulin is about 40:1 to about 60:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 1:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 2:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 3:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 4:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 5:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 6:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 7:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 8:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 9:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 10:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 11:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 12:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 13:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 14:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 15:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 16:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 17:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 18:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 19:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 20:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 25:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 30:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 35:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 40:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 45:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 50:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 60:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 70:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 80:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 90:1. In some embodiments, the ratio of a functional agent or an acyl donor substrate:immunoglobulin can be 100:1.

The contacting can be performed in a number of suitable conjugation buffers including, for example, DPBS, 1×PBS, pH 7.2, sodium phosphate, potassium phosphate, sodium borate, Tris, and HEPES, to name a few. The concentration of conjugation buffer include from about 5 mM to about 2 M, from about 5 mM to about 1 M, from about 5 mM to about 500 mM, from about 5 mM to about 100 mM, from about 10 mM to about 100 mM, from about 20 mM to about 100 mM, from about 30 mM to about 100 mM, from about 45 mM to about 100 mM, from about 60 mM to about 100 mM, from about 75 mM to about 100 mM, from about 10 mM to about 90 mM, from about 10 mM to about 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about 45 mM, or from about 10 mM to about 30 mM. In some embodiments, the concentration of the conjugation buffer can be about 10 mM. In some embodiments, the concentration of the conjugation buffer can be about 20 mM. In some embodiments, the concentration of the conjugation buffer can be about 30 mM. In some embodiments, the concentration of the conjugation buffer can be about 40 mM. In some embodiments, the concentration of the conjugation buffer can be about 50 mM. In some embodiments, the concentration of the conjugation buffer can be about 60 mM. In some embodiments, the concentration of the conjugation buffer can be about 70 mM. In some embodiments, the concentration of the conjugation buffer can be about 80 mM. In some embodiments, the concentration of the conjugation buffer can be about 90 mM. In some embodiments, the concentration of the conjugation buffer can be about 100 mM. In some embodiments, the concentration of the conjugation buffer can be about 250 mM. In some embodiments, the concentration of the conjugation buffer can be about 500 mM. In some embodiments, the concentration of the conjugation buffer can be about 750 mM. In some embodiments, the concentration of the conjugation buffer can be about 1 M. In some embodiments, the concentration of the conjugation buffer can be about 1.25 M. In some embodiments, the concentration of the conjugation buffer can be about 1.5 M. In some embodiments, the concentration of the conjugation buffer can be about 1.75 M. In some embodiments, the concentration of the conjugation buffer can be about 2 M.

The conjugation buffer can further include sodium chloride. Suitable concentrations of sodium chloride include from about 0 mM to about 2 M, from about 0 mM to about 1 M, from about 1 M to about 2 M, from about 500 mM to about 1.5 M, from about 25 mM to about 500 mM, from about 50 mM to about 500 mM, from about 75 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 200 mM to about 500 mM, from about 250 mM to about 500 mM, from about 300 mM to about 500 mM, from about 350 mM to about 500 mM, from about 400 mM to about 500 mM, from about 0 mM to about 400 mM, from about 0 mM to about 350 mM, from about 0 mM to about 300 mM, from about 0 mM to about 250 mM, from about 0 mM to about 200 mM, from about 0 mM to about 150 mM, from about 0 mM to about 100 mM, from about 0 mM to about 50 mM, or from about 0 mM to about 25 mM. In some embodiments, the concentration of sodium chloride can be about 25 mM. In some embodiments, the concentration of sodium chloride can be about 50 mM. In some embodiments, the concentration of sodium chloride can be about 75 mM. In some embodiments, the concentration of sodium chloride can be about 100 mM. In some embodiments, the concentration of sodium chloride can be about 150 mM. In some embodiments, the concentration of sodium chloride can be about 200 mM. In some embodiments, the concentration of sodium chloride can be about 250 mM. In some embodiments, the concentration of sodium chloride can be about 300 mM. In some embodiments, the concentration of sodium chloride can be about 350 mM. In some embodiments, the concentration of sodium chloride can be about 400 mM. In some embodiments, the concentration of sodium chloride can be about 500 mM. In some embodiments, the concentration of sodium chloride can be about 750 mM. In some embodiments, the concentration of sodium chloride can be about 1 M. In some embodiments, the concentration of sodium chloride can be about 1.25 M. In some embodiments, the concentration of sodium chloride can be about 1.5 M. In some embodiments, the concentration of sodium chloride can be about 1.75 M. In some embodiments, the concentration of sodium chloride can be about 2 M.

The pH of the conjugation buffer can be from about 4 to about 9. In some embodiments, the pH of the conjugation buffer can be about 5 to about 8. In another embodiment, the pH of the conjugation buffer can be about 6 to about 7. In some embodiments, the pH of the conjugation buffer can be about 4. In some embodiments, the pH of the conjugation buffer can be about 4.5. In some embodiments, the pH of the conjugation buffer can be about 5. In some embodiments, the pH of the conjugation buffer can be about 5.5. In some embodiments, the pH of the conjugation buffer can be about 6.0. In some embodiments, the pH of the conjugation buffer can be about 6.5. In some embodiments, the pH of the conjugation buffer can be about 6.6. In some embodiments, the pH of the conjugation buffer can be about 6.7. In some embodiments, the pH of the conjugation buffer can be about 6.8. In some embodiments, the pH of the conjugation buffer can be about 6.9. In some embodiments, the pH of the conjugation buffer can be about 7.0. In some embodiments, the pH of the conjugation buffer can be about 7.1. In some embodiments, the pH of the conjugation buffer can be about 7.2. In some embodiments, the pH of the conjugation buffer can be about 7.3. In some embodiments, the pH of the conjugation buffer can be about 7.4. In some embodiments, the pH of the conjugation buffer can be about 7.5. In some embodiments, the pH of the conjugation buffer can be about 7.6. In some embodiments, the pH of the conjugation buffer can be about 7.7. In some embodiments, the pH of the conjugation buffer can be about 7.8. In some embodiments, the pH of the conjugation buffer can be about 7.9. In some embodiments, the pH of the conjugation buffer can be about 8.0. In some embodiments, the pH of the conjugation buffer can be about 8.1. In some embodiments, the pH of the conjugation buffer can be about 8.2. In some embodiments, the pH of the conjugation buffer can be about 8.3. In some embodiments, the pH of the conjugation buffer can be about 8.4. In some embodiments, the pH of the conjugation buffer can be about 8.5. In some embodiments, the pH of the conjugation buffer can be about 9.

To facilitate solubility of a functional agent or an acyl donor substrate in the conjugation buffer, a final concentration of organic, water-miscible solvent in the conjugation buffer may be from about 0% to about 20%, from about 2% to about 20%, from about 5% to about 20%, from about 8% to about 20%, from about 11% to about 20%, from about 16% to about 20%, from about 0% to about 18%, from about 0% to about 15%, from about 0% to about 12%, from about 0% to about 10%, from about 0% to about 8%, from about 0% to about 6%, or from about 0% to about 2%.

The conjugation buffer can further comprise propylene glycol to facilitate solubility of the thiol-reactive compound in the conjugation buffer. Suitable concentrations of propylene glycol include from about 1% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%. In some embodiments, the concentration of propylene glycol can be about 1% or about 5%. In some embodiments, the concentration of propylene glycol can be about 10%. In some embodiments, the concentration of propylene glycol can be about 20%. In some embodiments, the concentration of propylene glycol can be about 30%. In some embodiments, the concentration of propylene glycol can be about 40%. In some embodiments, the concentration of propylene glycol can be about 50%.

The conjugation buffer can further comprise a non-ionic detergent to facilitate solubility of the conjugated immunoglobulin in the conjugation buffer. Exemplary non-ionic detergents include, but are not limited to, polysorbate-20 or polysorbate-80. Suitable concentrations of non-ionic detergent include from about 0% to about 1%, from about 0.1% to about 1%, from about 0.3% to about 1%, from about 0.5% to about 1%, from about 0.7% to about 1%, from about 0% to about 0.8%, from about 0% to about 0.6%, from about 0% to about 0.4%, or from about 0% to about 0.2%. In some embodiments, the concentration of non-ionic detergent can be about 0.1%. In some embodiments, the concentration of non-ionic detergent can be about 0.2%. In some embodiments, the concentration of non-ionic detergent can be about 0.3%. In some embodiments, the concentration of non-ionic detergent can be about 0.4%. In some embodiments, the concentration of non-ionic detergent can be about 0.5%. In some embodiments, the concentration of non-ionic detergent can be about 0.6%. In some embodiments, the concentration of non-ionic detergent can be about 0.7%. In some embodiments, the concentration of non-ionic detergent can be about 0.8%. In some embodiments, the concentration of non-ionic detergent can be about 0.9%. In some embodiments, the concentration of non-ionic detergent can be about 1.0%.

The contacting can be performed for about 30 minutes to about 48 hours, for about 1 hour to about 48 hours, for about 2 hours to about 24 hours, for about 24 hours to about 48 hours, for about 30 hours to about 48 hours, for about 36 hours to about 48 hours, for about 42 hours to about 48 hours, for about 2 hours to about 42 hours, for about 2 hours to about 36 hours, for about 2 hours to about 30 hours, for about 2 hours to about 24 hours, for about 2 hours to about 18 hours, for about 2 hours to about 12 hours, about 30 minutes to about 1 hour, about 30 minutes to about 2 hours, or for about 2 hours to about 6 hours. In some embodiments, the contacting can be performed for about 30 minutes. In some embodiments, the contacting can be performed for about 1 hour. In some embodiments, the contacting can be performed for about 1.5 hours. In some embodiments, the contacting can be performed for 2 hours. In some embodiments, the contacting can be performed for 6 hours. In some embodiments, the contacting can be performed for 12 hours. In some embodiments, the contacting can be performed for 18 hours. In some embodiments, the contacting can be performed for 24 hours. In some embodiments, the contacting can be performed for 30 hours. In some embodiments, the contacting can be performed for 36 hours. In some embodiments, the contacting can be performed for 42 hours. In some embodiments, the contacting can be performed for 48 hours.

The temperature of the contacting can be from about 4° C. to about 50° C., from about 18° C. to about 37° C., from about 20° C. to about 37° C., from about 22° C. to about 37° C., from about 24° C. to about 37° C., from about 26° C. to about 37° C., from about 28° C. to about 37° C., from about 30° C. to about 37° C., from about 32° C. to about 37° C., from about 34° C. to about 37° C., from about 18° C. to about 34° C., from about 18° C. to about 32° C., from about 18° C. to about 30° C., from about 18° C. to about 28° C., from about 18° C. to about 26° C., or from about 18° C. to about 24° C. In some embodiments, the contacting can be performed at 4° C. In some embodiments, the contacting can be performed at 18° C. In some embodiments, the contacting can be performed at 20° C. In some embodiments, the contacting can be performed at 22° C. In some embodiments, the contacting can be performed at 24° C. In some embodiments, the contacting can be performed at 26° C. In some embodiments, the contacting can be performed at 28° C. In some embodiments, the contacting can be performed at 30° C. In some embodiments, the contacting can be performed at 32° C. In some embodiments, the contacting can be performed at 34° C. In some embodiments, the contacting can be performed at 37° C. In some embodiments, the contacting can be performed at 50° C.

Unincorporated functional agent or acyl donor substrate can be separated from the conjugated immunoglobulin by desalting chromatography using a number of suitable resins including, but not limited to, G-25 resin, G-50 resin, Biogel P10, or other resins with exclusion limits of ranges 5,000-10,000 Da. Chromatography can be performed in column format or spin-column format, depending on scale. Suitable buffers for desalting include, for example, DPBS, 1×PBS, sodium phosphate, potassium phosphate, sodium borate, Tris, or HEPES-based buffers may substitute for 1×PBS.

In a first embodiment, the functional agent comprising an acyl donor substrate which comprises a glutamine residue conjugated to the engineered lysine residue via the acyl donor substrate. In this first embodiment, the functional agent is combined with the acyl donor substrate prior to conjugation with the immunoglobulin by reacting the reactive group on the acyl donor substrate with the functional agent. In a second embodiment, the acyl donor substrate comprising a glutamine residue and a reactive group is first conjugated to the immunoglobulin, and then the reactive group is joined to a functional agent.

The acyl donor substrates can comprise a linker, “L”. Linkers can be non-cleavable linkers or cleavable linkers. Exemplary linkers include, for example, disulfide containing linkers, acetal-based linkers, and ketal-based linkers. In some aspects, the linker can be a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, one or more amino acid, polyethylene glycol (PEG) or an alkyl. In some embodiments, the linker can comprise PEG. In some aspects, the linker can be a cleavable linker. Suitable cleavable linkers include, for example, valine-citrulline-para aminobenzyl. In some aspects, the linker can be a disulfide containing linker. In some aspects, the linker can be an acetal-based linker. In some aspects, the linker can be a ketal-based linker. A linker may also be one or more amino acids, alone or in combination with another linker such as one or more PEG groups.

The acyl donor substrate comprising a glutamine residue can be present in, part of, or attached to, a functional agent. Suitable functional agents include, for example, fluorophores, fluorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelators, lipids, nucleic acids (such as DNA or RNA) and drugs. In some aspects, the functional agent can comprise a fluorophore. In some aspects, the functional agent can comprise a fluorescent dye. In some aspects, the functional agent can comprise a polypeptide. In some aspects, the functional agent can comprise an immunoglobulin. In some aspects, the functional agent can comprise an antibiotic. In some aspects, the functional agent can comprise a nucleic acid (such as DNA or RNA). In some aspects, the functional agent can comprise a radionuclide. In some aspects, the functional agent can comprise a small molecule. In some aspects, the functional agent can comprise a chelator (for example, DOTA, CHX-A″-DTPA, NOTA, among others). In some aspects, the functional agent can comprise a lipid. In some aspects, the functional agent can comprise a drug. In some aspects, the functional agent can comprise a combination of any of the above listed functional agents.

The acyl donor substrate (i.e., a first acyl donor substrate) can be bound to a second acyl donor substrate or linker, the second acyl donor substrate or linker being bound to a second immunoglobulin having a second heavy chain variable region and a second light chain variable region, the second heavy chain variable region having an engineered lysine residue. For example, the first acyl donor substrate and the second acyl donor substrate can have a first and second chemical linker as the first and second functional agents, respectively. The first and second chemical linkers can be bound to each other by a number of suitable means including, for example, by click chemistry.

In one embodiment, the functional agent comprising an acyl donor substrate is according to one of formulae (I) or (II):

(Z)_(m)-Gln-(L)_(n)-(Y)  (I)

(Y)-(L)_(n)-Gln-(Z)_(m)  (II)

wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and Y is a functional agent.

In another embodiment, the acyl donor substrate is according to one of formulae (III) or (IV):

(Z)_(m)-Gln-(L)_(n)-(X)  (III)

(X)-(L)_(n)-Gln-(Z)_(m)  (IV)

wherein

Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and X is a reactive group.

In one embodiment, Z is a CBZ group. In another embodiment, Z is an amino acid residue.

In one embodiment, L is an amino acid residue. In one embodiment, n is 2-5, and each L is independently an amino acid residue. In another embodiment, L is a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted. In another embodiment, L is a polyethylene glycol (PEG) moiety. In another embodiment, n is 2-5, and one or more L comprises one or more amino acids and one or more additional L groups comprises a polyethylene glycol moiety (PEG).

In one embodiment, m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4. In another embodiment, m is 5.

In one embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5.

In one embodiment, X is (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN). In another embodiment, X is

(dibenzocyclooctyne; DBCO). In another embodiment, X is trans-cyclooctene (TCO). In another embodiment, X is azido (N₃). In another embodiment, X is alkyne. In another embodiment, X is tetrazine methylcyclopropene. In another embodiment, X is norbornene. In another embodiment, X is hydrazide/hydrazine. In another embodiment, X is aldehyde.

In one embodiment, for an acyl donor substrate according to formula (I), Z is a CBZ group; L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and n is 0, 1, 2 or 3.

In another embodiment, the acyl donor substrate is according to formula (I), wherein Z is a CBZ group, and L is an amino acid. In one embodiment, L is Gly. In one aspect of this embodiment, m is 1, and n is 1.

In one embodiment, the acyl donor substrate is according to formula (II), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one Lisa Gly.

In another embodiment, the functional agent Y is auristatin F.

In one embodiment, for an acyl donor substrate according to formula (III), Z is a CBZ group; L is a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and n is 0, 1, 2 or 3.

In another embodiment, the acyl donor substrate is according to formula (III), wherein Z is a CBZ group, and L is an amino acid. In one embodiment, L is Gly. In one aspect of this embodiment, m is 1, and n is 1.

In one embodiment, the acyl donor substrate is according to formula (IV), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is a Gly. In another embodiment, the functional agent Y is auristatin F.

The disclosed methods can be performed on a humanized immunoglobulin, or antigen-binding portion thereof. Thus, in some embodiments, the immunoglobulin, or antigen-binding portion thereof can be a humanized immunoglobulin, or antigen-binding portion thereof.

The disclosed methods can be performed on a human immunoglobulin, or antigen-binding portion thereof. Thus, in some embodiments, the immunoglobulin, or antigen-binding portion thereof can be a human immunoglobulin, or antigen-binding portion thereof. In another embodiment, the immunoglobulin, or antigen-binding portion thereof can be a non-human immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the disclosed methods can be performed on an IgG₁, IgG₂, IgG₃ or IgG₄ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgG₁ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgG₂ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgG₃ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgG₄ immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the disclosed methods can be performed on an IgA₁, IgA₂, or IgM immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgA₁ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgA₂ immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgM immunoglobulin, or antigen-binding portion thereof. In one embodiment, the IgA or IgM immunoglobulin, or antigen-binding portion thereof has a tail piece. In another embodiment, the IgA or IgM immunoglobulin, or antigen-binding portion thereof has the tail piece removed.

In one embodiment, the method is performed on an IgD or IgE immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgD immunoglobulin, or antigen-binding portion thereof. In one embodiment, the method is performed on an IgE immunoglobulin, or antigen-binding portion thereof.

For the methods described herein, in one embodiment, the microbial transglutaminase is from Actinomadura sp. T-2, Bacillus circulans BL32, Bacillus subtilis spores, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Enterobacter sp. C2361, Providencia sp. C1112, Streptoverticillium mobaraense (aka Streptomyces mobaraensis), Streptomyces platensis M5218, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces lividans JT46/pAE053, Streptomyces lydicus, Streptomyces platensis, Streptomyces sioyansis, Streptoverticillium griseocarneum, Streptoverticillium ladakanum NRRL-3191, Streptoverticillium sp. s-8112, or Streptococcus suis. In one embodiment, the microbial transglutaminase is from Streptomyces mobaraensis.

For the methods described herein, in one embodiment, the transglutaminase is isolated from a plant selected from the group consisting of Medicago sativa, Beta vulgaris, Helianthus tuberosus, Zea mays, Glycine max, Arabidopsis thaliana, Nicotiana tabacum, Chlamydomonas reinhardtii, Dunaliella salina, Oryza sativa, and Rosmarinus officinalis L.

For the methods described herein, in one embodiment, the transglutaminase is mammalian and is isolated from Transglutaminase 1 thru 7 and Factor XIII

In one embodiment, the transglutaminase is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a microbial transglutaminase described herein. In one embodiment, the transglutaminase is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the microbial transglutaminase is from Streptomyces mobaraensis. Transglutaminase enzymes can be purchased from Ajinomoto® or Zedira (Product number T001). In another embodiment, the transglutaminase is purified. In another embodiment, the transglutaminase is recombinantly expressed and subsequently purified using methods known to one of ordinary skill in the art.

In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 0.1 units/mL to about 250 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 1 unit/mL to about 25 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 1 unit/mL to about 25 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 0.1 unit/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 0.5 unit/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 1 unit/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 5 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 10 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 15 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 20 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 25 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 50 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 75 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 100 units/mL. In one embodiment, the transglutaminase enzyme is present in the methods described herein in a concentration of about 150 units/mL, 200 units/mL, or 250 units/mL.

For the methods provided herein, in one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 200:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 100:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 25:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 20:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 15:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 10:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 9:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 8:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 7:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 6:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 5:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 4:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 3:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 2:1. In one embodiment, the ratio of functional agent to immunoglobulin is about 1:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 10:1 to about 100:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 50:1 to about 200:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 50:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 100:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 1:1 to about 150:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 50:1 to about 100:1. In one embodiment, the ratio of functional agent to immunoglobulin is from about 100:1 to about 200:1.

In one embodiment, the ratio of functional agent to immunoglobulin is about 20:1. In one embodiment, the ratio of functional agent to immunoglobulin is known and is consistently reproducible by following the methods disclosed herein. In some embodiments, the ratio of a functional agent:immunoglobulin is about 1:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 2:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 3:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 4:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 5:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 6:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 7:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 8:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 9:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 10:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 11:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 12:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 13:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 14:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 15:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 16:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 17:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 18:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 19:1. In some embodiments, the ratio of a functional agent:immunoglobulin is about 20:1. The ratio of functional agent to immunoglobulin, as used herein, is calculated based on an average of the conjugation ratio of the functional agent to an immunoglobulin in a pool of antibodies in a composition.

In embodiments of the present invention, acyl acceptor sites are engineered in a immunoglobulin, or antigen-binding portion thereof, by either insertion of a lysine between two natural amino acid residues, or by a the substitution of a natural amino acid with lysine substitution. Engineered lysine residues may be located at one or more positions throughout the sequence of an immunoglobulin. The optimal context for an acyl acceptor site is not only position-dependent, but also requires that the amino acid residue adjacent to and immediately before the engineered lysine not be an acidic amino acid residue; and that the amino acid residue adjacent to and immediately after the engineered lysine not be an acidic amino acid residue or a proline residue. MTGase conjugation technology can utilize these engineered sites to conjugate a variety of acyl donor-containing functional agents for making antibody drug conjugates (ADCs), bispecific antibodies, immunotoxins, or other mAb-protein complexes. In another embodiment, combining multiple engineered acyl acceptor sites, the theoretical drug-to-antibody ratio (DAR) increases for each engineered acyl acceptor site. Increasing the DAR of an ADC results in delivery of more functional agents into a subject per monoclonal antibody, which allows for defined DARs, better product homogeneity, and lower patient dosing.

In embodiments provided herein, wherein at least two additional engineered lysine residues are present, the ratio of functional agent to immunoglobulin is increased based on the number of additional engineered lysine residues. For example, wherein two engineered lysine residues are present, resulting in an antibody with four transamidation sites and a ratio of functional agent to immunoglobulin of about 2:1 to about 4:1. As another example, wherein three engineered lysine residues are present, resulting in an antibody with six transamidation sites and a ratio of functional agent to immunoglobulin of about 2:1 to about 6:1.

Conjugated Immunoglobulins

Also disclosed herein are conjugated immunoglobulins comprising any of the immunoglobulins or antigen-binding portions thereof disclosed herein, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, and is conjugated to a functional agent comprising an acyl donor substrate, wherein the acyl donor substrate comprises a glutamine residue. Additional embodiments include conjugated immunoglobulins comprising any of the immunoglobulins or antigen-binding portions thereof disclosed herein, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, and is conjugated to an acyl donor substrate, wherein the acyl donor substrate comprises a glutamine residue and a reactive group, wherein the reactive group can be reacted with a functional agent after the conjugation of the acyl donor substrate to the immunoglobulin, or antigen-binding portion thereof.

In one embodiment, the amino acid residues flanking the engineered lysine are also engineered, e.g., mutated to optimize the sequence for an engineered acyl acceptor lysine site on an immunoglobulin. For example, the amino acid residue adjacent to and immediately after the engineered lysine residue (e g, amino acid position +1) may be mutated to any amino acid residue other than proline or an acidic amino acid residue. The amino acid residue adjacent to and immediately before the engineered lysine residue (e.g., amino acid position −1) may be mutated to any amino acid residue other than an acidic amino acid residue.

In one embodiment, the amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) may be mutated to lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is glycine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is lysine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is alanine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is valine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is leucine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is isoleucine. In one embodiment, the mutated amino acid residue adjacent to and immediately after to the engineered lysine (amino acid position +1) is methionine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is phenylalanine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is tyrosine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is tryptophan. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is serine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is threonine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is cysteine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is asparagine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is glutamine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is histidine. In one embodiment, the mutated amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is arginine. In one embodiment, wherein the amino acid residue adjacent to and immediately after the engineered lysine (amino acid position +1) is not proline, aspartic acid, or glutamic acid.

In one embodiment, the amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) may be mutated to lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is glycine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is lysine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is alanine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is valine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is leucine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is isoleucine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is methionine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is phenylalanine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is tyrosine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is tryptophan. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is serine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is threonine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is cysteine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is asparagine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is glutamine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is histidine. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is proline. In one embodiment, the mutated amino acid residue adjacent to and immediately before the engineered lysine (amino acid position −1) is arginine. In one embodiment, wherein the amino acid residue adjacent to and immediately before the engineered lysine (amino acid position +1) is not aspartic acid or glutamic acid.

In some embodiments, the immunoglobulin can be humanized. In other embodiments, the immunoglobulin is human. In another embodiment, the immunoglobulin is chimeric.

The acyl donor substrate comprising a glutamine residue and a reactive group can also comprise a linker, “L”. Likewise, the functional agents which contain an acyl donor substrate comprising a glutamine residue can have a linker between the functional agent and the acyl donor substrate portion of the molecule. Linkers can be non-cleavable linkers or cleavable linkers. Exemplary linkers include, for example, disulfide containing linkers, acetal-based linkers, and ketal-based linkers. In some aspects, the linker can be a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, polyethylene glycol (PEG) or an alkyl. In some embodiments, the linker can comprise PEG. In some aspects, the linker can be a cleavable linker. Suitable cleavable linkers include, for example, valine-citrulline-para aminobenzyl. In some aspects, the linker can be a disulfide containing linker. In some aspects, the linker can be an acetal-based linker. In some aspects, the linker can be a ketal-based linker.

The conjugated immunoglobulins of the invention comprise a functional agent. Suitable functional agents include, for example, a therapeutic agent or a diagnostic agent. Suitable functional agents include, for example, fluorophores, fluorescent dyes, polypeptides, immunoglobulins, antibiotics, nucleic acids, radionuclides, chemical linkers, small molecules, chelators, lipids, and drugs. In some aspects, the functional agent can comprise a fluorophore. In some aspects, the functional agent can comprise a fluorescent dye. In some aspects, the functional agent can comprise a polypeptide. In some aspects, the functional agent can comprise an immunoglobulin. In some aspects, the functional agent can comprise an antibiotic. In some aspects, the functional agent can comprise a nucleic acid (such as DNA or RNA). In some aspects, the functional agent can comprise a radionuclide. In some aspects, the functional agent can comprise a small molecule. In some aspects, the functional agent can comprise a chelator (for example, DOTA, CHX-A″-DTPA, NOTA, among others). In some aspects, the functional agent can comprise a lipid. In some aspects, the functional agent can comprise a drug. In some aspects, the functional agent can comprise a combination of any of the above listed functional agents.

Accordingly, the disclosed conjugated immunoglobulins include, but are not limited to, immunoglobulin-fluorophore conjugates, immunoglobulin-fluorescent dye conjugates, immunoglobulin-polypeptide conjugates, immunoglobulin-immunoglobulin conjugates, immunoglobulin-antibiotic conjugates, immunoglobulin-nucleic acid conjugates, immunoglobulin-radionuclide conjugates, immunoglobulin-chemical linker conjugates, immunoglobulin-small molecule conjugates, immunoglobulin-chelator conjugates, immunoglobulin-lipid conjugates, and immunoglobulin-drug conjugates.

Any of the immunoglobulins disclosed herein can be conjugated to any of the functional agents disclosed herein. For example, the conjugated immunoglobulin can comprise a fluorophore, fluorescent dye, polypeptide, immunoglobulin, antibiotic, nucleic acid, radionuclide, chemical linker, small molecule, chelator, lipid, or drug.

In some embodiments, the immunoglobulin can be conjugated to a small molecule antineoplastic agent, such as an auristatin. In some aspects, the functional agent can be auristatin F (AuF). Thus, the disclosed conjugated immunoglobulins include any of the above disclosed immunoglobulins conjugated to auristatin F (AuF-T135K conjugate).

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions can comprise any of the immunoglobulins disclosed herein. In some embodiments, the pharmaceutical compositions can comprise any of the conjugated immunoglobulins disclosed herein. In one embodiment, the pharmaceutical composition comprises the conjugated immunoglobulin and a pharmaceutically acceptable carrier.

Nucleic Acid Molecules Encoding Conjugatable Immunoglobulins and Host Cells Comprising the Same

Also provided herein are nucleic acid molecules encoding any of the conjugatable immunoglobulins or antigen-binding portions thereof disclosed herein. As an example, in one embodiment, the nucleic acid molecule encodes a conjugatable immunoglobulin comprising a heavy chain variable region and a light chain variable region and wherein the encoded conjugatable immunoglobulin comprises an engineered lysine residue, and wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue. The engineered lysine residue of the immunoglobulin or antigen-binding portions thereof, is conjugated to the glutamine residue of an acyl donor substrate, thereby generating the conjugated immunoglobulin.

Also disclosed are host cells comprising any of the disclosed nucleic acid molecules or plasmids that encode the conjugatable immunoglobulin or antigen-binding portions thereof. Suitable host cells include, but are not limited to, mammalian cells, bacterial cells, yeast cells, insect cells, to name a few.

The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.

EXAMPLES Example 1: Materials & Methods Site-Directed Mutagenesis

Mutations were generated using Stratagene's QuikChange XL according to the manufacturer's protocol. The desired mutations were confirmed by DNA sequencing.

Deletion Mutagenesis

HC fragments for Fab expression were made by PCR amplifying the HC leader sequence through the hinge that terminated at various 3′ codons. The PCR fragments were cloned into a pcDNA3.1-based mammalian expression plasmid using an In-Fusion HD cloning kit according to the manufacturer's protocol (CLONTECH).

Transfection and Stable Cell Line Generation

For each milliliter of cells to be transfected with ExpiFectamine, 333.3 ng HC plasmid and 333.3 ng LC plasmid was contacted for 5-10 min in 50 μL Opti-MEM (ThermoFisher Scientific, Waltham, Mass.). Likewise, 2.67 μL ExpiFectamine was contacted in 50 μL Opti-MEM. The ExpiFectamine solution was added to the DNA mixture, and incubated for 20-30 min at room temperature. The DNA:ExpiFectamine mixture was added to the cells while swirling and incubated at 37° C., 8% CO₂, shaking at 125 rpm. The following day, 5 μL of enhancer 1 and 50 μL of enhancer 2 per mL of cells were added to the transfection with continued incubation for another 7-10 days.

Antibody-expressing stable pools were selected by adding 1 mL of transfectants to 14 mL DMEM in a T75 flask with 5 μg/mL blasticidin and 400 μg/mL zeocin (Invivogen, San Diego, Calif.) one to three days after transfection. After drug-resistant cells grew to confluency, the medium was replaced with FreeStyle 293 expression medium for 24 to 48 h. Cells were physically dislodged by tapping the flask (trypsinization resulted in low viability, data not shown) and were then seeded at 6×10⁵ cells/mL in 30 mL FreeStyle 293 expression medium in a 125-mL shake flask. Cultures were incubated at 37° C. in 8% CO₂ with shaking at 125 rpm.

MAb and Fab Production

Stably-transfected cell line pools were seeded at 0.6 to 1×10⁶ cells/mL in FreeStyle 293 expression medium. Cells were incubated at 37° C., 8% CO₂, shaking at 125 rpm. Two days after the culture reached a density of 1×10⁶ cells/mL, cultures were fed with final concentrations of 10 g/L Select Soytone (BD Biosciences, San Jose, Calif.), 5 mM valeric acid (Sigma Aldrich, St. Louis, Mo.), and 1:100 CD Lipid Concentrate (ThermoFisher Scientific, Waltham, Mass.). When the cell viability was less than 50% (7-10 days), the cultures were centrifuged for 1 h at 8000 rpm in a Beckman JLA8.1000 rotor. The supernatant was then filtered through a 0.2 μm PES filter and stored at 4° C. or −20° C. until purification.

MAb and Fab Purification

MAbs were purified using one of two methods. For mAb and Fab supernatants less than 10 mL, affinity chromatography was performed using a batch purification method with protein A resin or anti-kappa resin, respectively. MAb and Fab supernatants greater than 25 mL were purified using pre-packed protein A or anti-kappa columns, respectively.

Batch Purification

Prosep-vA High Capacity Protein A resin (Millipore, Billerica, Mass.) was equilibrated with DPBS, and 100 μL were added to 3 to 6 mL of sample. Following incubation at 4° C. for 1 hour to overnight, the resin was washed three times with 1 mL DPBS and centrifuged at 18,000×g for 30 s. The sample was eluted from the resin by addition of 400 μL 0.1 M Glycine, pH 2.9 followed by centrifugation at 18,000×g for 30 s. The sample was neutralized with 40 μL of 1 M Tris, pH 8.0. The buffer was exchanged using 0.5 mL Amicon Ultra, 10k cutoff filters (Millipore, Billerica, Mass.) by concentrating the sample to 100 μL by centrifugation at 18,000×g for 3 to 5 minutes. The concentrated sample was diluted in 400 μL DPBS, followed by centrifugation. The process was repeated a total of four times.

Column Purification

A protein A or HiTrap KappaSelect column (GE Healthcare, Little Chalfont, UK) was equilibrated with 10 column volumes (CV) of 20 mM sodium phosphate, 10 mM EDTA, pH 7.2. The sample was then loaded, followed by washing unbound material with 10 CV of equilibration buffer. The sample was eluted using 5 CV of 0.1 M Glycine, pH 2.9. The fractions containing the mAb were pooled and dialyzed in DPBS using a MWCO 20K Slide-A-Lyzer (ThermoFisher Scientific, Waltham, Mass.).

Z-Gln-Gly Substrate Synthesis

Z-Gln-Gly was purchased from Bachem, and Z-Gln-Gly-CAD-biotin was purchased from ZEDIRA (FIG. 2).

Z-Gln-Gly-pentafluorophenyl ester (Z-Gln-Gly-PFP)

Synthesis was as described by Pasternack (Pasternack et al. 1997), with modifications (FIG. 3). Z-Gln-Gly (328.8 mg, 0.975 mmol) and pentafluorophenol (Sigma, 183.3 mg, 0.996 mmol) were dissolved in 10 mL NN′-dimethylformamide (DMF). EDAC-HCl (Sigma, 201 mg, 1.04 mmol) was then added and the reaction was incubated at room temperature under N₂ for 2 hr. 100 mL of cold diethyl ether was added to the reaction and precipitated overnight at −80° C. The crude product was collected by centrifugation and re-crystallized from 20 mL 60° C. methanol. The final product was rinsed with cold diethyl ether and dried over a stream of N₂. Final yield was 219.04 mg (44.7%). Electrospray ionization-mass spectrometry (ESI-MS) (direct infusion in 50% acetonitrile in 0.1% formic acid) m/z 504.0 ([M+H], 86%), 526.0 ([M+Na], 100%), 542.0 ([M+K], 22%).

Z-Gln-Gly-propyl azide (Z-Gln-Gly-N₃)

Z-Gln-Gly-PFP (21.24 mg, 4.22×10⁻⁵ mol) and azidopropylamine (Click Chemistry Tools, 42.2 μL of a 0.91 M stock solution in DMF, 3.84×10⁻⁵ mol) were dissolved in 0.42 mL final volume of DMF. Reaction was stirred under N₂ overnight at room temperature. Product was purified by HPLC using a 0.1% formic acid in H₂O/0.1% formic acid in acetonitrile mobile phase. Product was dried in vacuo. Final yield was 10.7 mg (60.4%). ESI-MS (gradient purification) m/z 420.2 ([M+H], 100%), 442.1 ([M+Na], 32%).

Z-Gln-Gly-PEG₂-bicyclononyne (Z-Gln-Gly-PEG₂-BCN)

Z-Gln-Gly-PFP (18.4 mg, 3.66×10⁻⁵ mol) and N-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (Sigma Aldrich) were dissolved in 0.37 mL final volume of DMF. Reaction was stirred under N₂ overnight at room temperature. Product was purified by HPLC using a 0.1% formic acid in H₂O/0.1% formic acid in acetonitrile mobile phase. Product was dried in vacuo. Final yield was 0.6 mg (2%). ESI-MS (gradient purification) m/z 688.2 ([M+H], 100%), 710.2 ([M+Na], 69%).

Z-Gln-Gly-PEG₂-Auristatin F (Z-Gln-Gly-PEG₂-AuF)

Z-Gln-Gly-PFP (22.2 mg, 4.37×10⁻⁵ mol) was dissolved in 0.85 mL DMF and 1,2-ethylenediamine (2.3×10⁻⁵ L, 3.5×10⁻⁴ mol) was added and mixed. Reaction was stirred under N₂ overnight at room temperature. Product was purified by HPLC using a 0.1% formic acid in H₂O/0.1% formic acid in acetonitrile mobile phase. Product was dried in vacuo. Final yield of Z-Gln-Gly-NH₂ was 3.8 mg (23%). ESI-MS (gradient purification) m/z 380.1 ([M+H], 100%). Z-Gln-Gly-NH₂ (3.8 mg, 1.01×10⁻⁵ mol) and NHS-PEG₂-AuF (10.3 mg, 1.03×10⁻⁵ mol) were dissolved in 0.2 mL DMF. Triethylamine (14 μL, 1×10⁻⁴ mol) was added and reaction was incubated under N₂ overnight at room temperature. Half of the reaction was purified by HPLC using a 0.1% formic acid in H₂O/0.1% formic acid in acetonitrile mobile phase. Product was dried in vacuo. Final yield of CBZ-Gln-Gly-PEG₂-AuF was 3.8 mg (60%). ESI-MS (gradient purification) m/z 634.0 ([M+H]²⁺,100%), 645.1 ([M+Na]²⁺,45%). 1267.0 ([M+H], 16%).

Microbial Transglutaminase Reaction

MAbs ranging in concentrations from 100 μg/mL to 2.5 mg/mL were contacted with 785 μM Z-Gln-Gly-biotin (Zedira, Darmstadt, Germany), Z-Gln-Gly-N₃, Z-Gln-Gly-PEG₂-BCN, or Z-Gln-Gly-PEG₂-AuF with 1 U/mL microbial transglutaminase (Zedira, Darmstadt, Germany) in DPBS for at least 16 h at 37° C.

High-Throughput mTGase Assay

MAbs ranging in concentrations from 500 ng/mL to 10 μg/mL were incubated with 60 μM Z-Gln-Gly-biotin and 0.1 U/mL mTGase (Zedira) in DPBS for at least 16 h at 37° C. A 96-well plate was coated with 1 μg/mL goat-anti-human IgG Fcγ mAb (Jackson ImmunoResesearch) overnight at 4° C. After washing the plate, the overnight mTGase reactions were diluted 1:10 in 50 μL DPBS, added to the plate, and incubated for 1 h at 22° C. The plate was then washed, and 0.1 μg/mL of streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResesearch) was added to the wells. The plate was washed again, and streptavidin-HRP-bound biotinylated samples were quantitated in relative fluorescent units (RFUs) using QuantaBlue substrate (Thermo) according to the manufacturer's protocol.

Ultra-Performance Liquid Chromatography (UPLC)/ESI-MS Analysis of mAb Conjugation

Purified antibodies were diluted to 1 mg/mL in DPBS (if below 1.0 mg/mL samples were left at original concentration). Reactions containing dimethylsulfoxide (DMSO) were desalted using a Zeba spin desalting column. The mAbs were then either deglycosylated using PNGase F (NEB) or digested into Fab′₂ and Fc fragments by IdeS (Promega). To deglycosylate the mAbs, G7 buffer (5 or 10 μL) and PNGase F (1 or 2 μL) were added to the mAb (50 or 100 μL). The reaction was incubated in a Discover microwave (CEM) for 2 cycles: 1.) microwave power 10 W, 37° C., 10 min, and then wait for 3-5 min; 2.) microwave power 2 W, 37° C., 10 min. A portion of the deglycosylated sample was reduced by adding dithiothreitol (DTT) to a final concentration of 20 mM, followed by incubation at 60° C. for 3 min. To generate Fab′₂ and Fc fragments, 50 U/μL of IdeS was added to 0.5 mg/mL of mAb and incubated at 37° C. for 0.5-1 h. The IdeS samples were or were not reduced.

Samples were then analyzed using a Waters Acquity UPLC and Q-Tof Premier mass spectrometer. Samples (0.5-2 μg each) were injected onto a MassPrep micro desalting column at 65° C., eluted from the column with a 5 min equilibration in 95% of mobile phase A, a 10 min gradient (5-90% B), and a 10 min re-equilibration in 95% of mobile phase A, at 0.05 mL/min Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetonitrile. The Q-Tof mass spectrometer was run in positive ion, V-mode with detection in the range of 500-4000 m/z. The source parameters were as follows: capillary voltage, 2.25 kV (intact antibody)-2.50 kV (reduced antibody); sampling cone voltage, 65.0 V (intact antibody) or 50.0 V (reduced antibody); source temperature, 100° C.; desolvation temperature, 250° C.; desolvation gas flow, 550 L/hr. The protein peak was deconvoluted using the MassLynx MaxEnt 1 function.

Reverse Phase Liquid Chromatography (LC)-MS

Samples were analyzed using reverse phase liquid chromatography. Samples containing 100 μL of ADC at the concentration of 1-2 mg/mL were reduced with 20 mM DTT at 60° C. for 3 minutes. The samples were analyzed using Waters Alliance HPLC with SQD and PDA detectors. Each sample was injected onto a Proteomix RP-1000 column (5μ, 4.6×150 mm, Sepax) at 65° C. For the LC and HC mutants, separation of the LC and HC occurred with a 3.0 min equilibration in 75% of mobile phase A (0.1% TFA in water) and a 27-minute gradient (25-55% mobile phase B [0.1% TFA in water]) at a flow rate of 1 mL/min

The SQD mass spectrometer was run in positive ion, V-mode with detection in the range of 200-2000 m/z. Source parameters were as follows: capillary voltage, 3.20 kV; sampling cone voltage, 40° C.; source temperature, 150° C.; desolvation temperature, 250° C.; desolvation gas flow, 700 L/hr. Scan time, 1 second. The protein peak was deconvoluted by the MassLynx MaxEnt 1 function. The PDA detector was at 280 nm.

The DAR was calculated based on the relative signal intensity of the unconjugated and conjugated LC and unconjugated and conjugated HC. Total DAR was calculated using the following equation: total DAR=(DAR LC+DAR HC)×2.

Example 2: Analysis of Solvent Exposed Lysines on IgG Antibodies

In order to further study the availability of solvent exposed lysines on IgG antibodies, the crystal structures of an IgG1-kappa Fab (4F3F), an IgG1-lambda Fab (4HK0), and IgG1 Fc (1FC1) were examined for potential acyl acceptor sites. As mTGase tends to prefer solvent-exposed substrate glutamines and lysines within loops (Spolaore et al. 2012), solvent exposed lysines were highlighted using Discovery Studio v4.5 with a 1.4 Å probe radius (FIG. 4). There are 7 solvent-exposed lysines in the Antibody 01 VH with 3 in turns or loops. As the number of lysines can vary between mAbs due to utilization of different germline variable region families and somatic hypermutation, the solvent exposure of lysines in the VH region of five other antibodies were also analyzed based on analogous positions of residues in the 4F3F structure. These VH regions potentially contain 1-5 solvent-exposed lysines with 1 or 2 present in a turn or loop. In the Antibody 01 Vκ there are 6 solvent exposed lysines and 4 are in loops or turns. The VK regions from four other antibodies potentially contain 3 to 5 solvent-exposed lysines with 2 in a loop or turn. Antibody 05 utilizes a lambda chain, and the solvent exposure of the lysines was determined using the crystal structure of 4HK0 based on sequence similarity of the light chain. Antibody 05 potentially has 2 solvent-exposed lysines in the Vλ domain with only 1 in a loop. The IgG1 constant domains have 23 solvent-exposed lysines with 13 in loops or turns (FIG. 6). The kappa constant region has 8 lysines with 5 in a turn or loop. The lambda has 6 solvent-exposed lysines with half in loops or turns. In total, the analyzed antibodies range from 42 to 50 solvent-exposed lysines in loops or turns per mAb.

To determine whether microbial transglutaminase can transamidate a native lysine residue on an IgG antibody, antibodies were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight. The samples were digested with IdeS and reduced with DTT, and the masses of the LC, Fd, and Fc fragments were analyzed by mass spectrometry. Two mass peaks corresponding to the GOF (+1445 Da) and G1F (+1608 Da) glycoforms were observed for each Fc. Antibody 04 also contained an N-linked glycosylation site in VH and two glycan species, and G2FS and G2FS2 glycans were observed. All samples lacked the C-terminal lysine (128 Da), as evidenced by the −130 to −132 Da difference between the observed and theoretical mass for the Fc. Although there are 42-50 potential acyl acceptor lysines in the different antibodies, surprisingly neither the HC nor the LC was modified by the acyl donor substrate (FIG. 7; Table 1).

TABLE 1 ESI-MS analysis of antibodies contacted with an acyl donor and microbial transglutaminase ZQG-CAD-biotin: +631 Da LC Fd Fc Calculated Observed ΔMass Calculated Observed Glycan ΔMass Calculated Glycan Observed ΔMass Antibody 23751 23750 −1 25073 25071 −2 25328 G0F 25198 −130 02 25491 G1F 25360 −131 Antibody 23478 23478 0 26097 26097 0 25388 G0F 25258 −130 03 25551 G1F 25420 −131 Antibody 23216 23213 −3 25072 25069 −3 25328 G0F 25198 −130 01 25491 G1F 25359 −132 Antibody 23532 23530 −2 27566 27564 G2FS −2 25296 G0F 25166 −130 04 27857 27855 G2FS2 −2 25459 G1F 25327 −132 Antibody 22655 22653 −2 26340 26337 −3 25328 G0F 25198 −130 05 25491 G1F 25360 −131 Antibody 23472 23470 −2 25383 25381 −2 25328 G0F 25198 −130 06 25491 G1F 25359 −132 Table 1: The masses of the LC, Fd, and Fc were determined by ESI-MS. The theoretical mass of each fragment was determined by the amino acid sequence subtracted from the observed mass to determine the change in mass (Δmass). A Δmass of −128 Da is due to cleavage of Lys447. The Fc is glycosylated with one or two oligosaccharides, G0F or G1F.

Example 3: Mutation of Natural Amino Acid Residues to Lysine to Create an Acyl Acceptor Site

Since there are no acyl acceptor sites in human IgG1 mAbs, lysine scanning mutagenesis was performed on Antibody 01 to identify regions that could be engineered to introduce an acyl acceptor site(s). In order to increase the chances of identifying novel acyl acceptor sites, mutagenesis was limited to solvent-exposed residues within the loops or turns of the constant regions of human IgG1 HC and kappa and lambda LCs (FIG. 6). Mutant mAbs were initially screened for transamidation using an ELISA-based assay by incubating mutants with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C. MAbs were captured on an anti-Fcγ coated plate and biotinylated mAbs were detected by HRP-conjugated streptavidin. Wild-type Antibody 01 and Antibody 01-L (which is transamidated at Lys447) were included as negative and positive controls, respectively. The signal for the positive control Antibody 01-L was greater than 12,000 RFU and mutant mAbs ranged from ˜1100 RFU to ˜11,000 RFU (FIG. 7).

Transamidation of all CH1 and upper hinge mutants to Z-Gln-Gly was analyzed By ESI-MS to determine whether the percentage of conjugation correlates with the RFU signal seen in the ELISA assay. Samples were incubated with mTGase and Z-Gln-Gly overnight at 37° C., and their masses were analyzed by ESI-MS. The RFU signal from the ELISA mostly correlated with the ESI-MS data where RFUs greater than 7000 corresponded to >70% conjugation of mutants S136K, D221K, T223K, and H224K (FIG. 7). The RFU signal for T135K and T225K was below 7000, but those mutants were 80.4% and 100% conjugated, respectively. In contrast, mutant G137K had an RFU of 8616, but only 38.9% was conjugated. Therefore, while a RFU signal >7000 corresponded to a high percentage of transamidation, there were a few false positives and false negatives. Rather than performing a low-throughput ESI-MS screen of the remaining mutants, a cutoff of >4000 RFU was used to pick CH2 and CH3 samples for analysis. Since only two kappa and no lambda samples were greater than 4000 RFU, samples greater than 3000 RFUs were also analyzed.

The transamidation reaction was performed by incubating Z-Gln-Gly-CAD-biotin and mTGase overnight at 37° C. with CH2 mutants M252K, E283K, A287K, and N297K; CH3 mutants P343K, G385K, G420K, H433K, L443K, S444K, and P445K; kappa mutants D151K, L201K, S202K, and E213K; and lambda mutants V147K, Q187K, and E213K, and subsequent analysis was performed by ESI-MS (FIG. 7). Three additional CH1 mutants, S191K, S192K, and L193K, were analyzed by ESI-MS, but were not including in the initial ELISA screen. Of these mutants, the CH1 mutant L193K, CH2 mutant M252K, the CH3 mutant P445K, the kappa mutants L201K and S202K, and the lambda mutant E213K were greater than 70% conjugated (FIG. 7). The results for the CH2 mutant N297K were inconclusive due to very low signal.

The mutants identified as having the highest conjugation efficiency were re-analyzed in a single experiment using the Z-Gln-Gly-CAD-biotin substrate. The region of conjugation was confirmed by digesting the samples with IdeS and reduction prior to ESI-MS analysis to generate Fd (CH1 and hinge), Fc (CH2 and CH3), and LC fragments. Conjugation to N297K was again inconclusive due to low Fc signal despite high Fd and LC signals. Transamidation of samples all correlated to the domain containing the lysine mutation. T135K and P445K mutants were conjugated at 100%, and conjugation to M252K was nearly 100% (FIG. 8). The S136K and S221K mutations were greater than 80% conjugated. The other three hinge mutants had conjugation efficiencies of less than 50%. Both LC mutants were greater than 80% conjugated.

Example 4: Inserting a Lysine Residue to Create an Acyl Acceptor Site

Residues Ser190 through Thr195 form a beta turn that connects beta strands E and F. Lysine scanning through this exposed area showed only one site—position 193—is an acceptable acyl donor site. This region forms an alpha helix, and it was possible that this secondary structure prevents transamidation in this region. To potentially disrupt the structure in this region, a lysine was inserted between Ser191 and Ser192, Ser192 and Leu193, or Leu193 and Gly194. Samples were incubated with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C., and their masses were analyzed by ESI-MS. Insertion of a lysine between Ser191 and Ser192 or Ser192 and Leu193 was 100% transamidated, but not when inserted between Leu193 and Gly194 (Table 2).

TABLE 2 (SEQ ID NOS: 295-297) Transamidation of a lysine insertion Z-Gln-Gly-CAD -biotin: +631 Da % sequence Calculated Observed ΔMass conjugated S191.K.S192 SSKSLGT 48937 49565 628 100.0% S192.K.L193 SSSKLGT 48937 49565 628 100.0% L193.K.G194 SSSLKGT 48937 48934  -3   0.0% MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight. The masses of the non-reduced mAbs were analyzed by ESI-MS (data not shown), and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as above. The DAR was determined by dividing the Δmass by the mass of Z-Gln-Gly-CAD-biotin.

Example 5: Transamidation Resulting From Addition of a Lysine to the LC C Terminus

It was previously demonstrated that the HC C-terminal Lys447 residue is a site of transamidation when cleavage of Lys447 is blocked by an additional C-terminal residue at position 448, see U.S. Provisional Application No. 62/269,138, filed on Dec. 18, 2015, and PCT/US2016/067165 filed on Dec. 16, 2016, the entire contents of each of which are expressly incorporated herein by reference. A lysine was engineered to the C terminus of the LC to determine whether a single C-terminal lysine extension is sufficient to act as an acyl acceptor site. In contrast to the native C-terminal lysine in the HC, a C-terminal lysine on the LC was not cleaved by a carboxypeptidase (Table 3). However, this engineered lysine was not an acyl acceptor site. An additional leucine was engineered to the C terminus of the engineered lysine (LC-KL), as a Lys-Leu motif at the C terminus of the HC is efficiently transamidated. However, LC-KL was not transamidated. The native C-terminal cysteine (Cys214) in the LC forms an interchain disulfide bond with the HC, and this region is buried in crystal structures of this region (data not shown). It could be that steric hindrance or lack of solvent exposure prevents transamidation. A single leucine was added between Cys214 and the lysine in an attempt to make the lysine more accessible to MTGase. Again, no cleavage was seen to the C-terminal lysine, and no transamidation was observed to the LK motif. However 9.1% of the LCs with the LKL motif were transamidated. To extend the lysine further away from the LC-HC interface, a Gly-Ser linker was inserted between Cys214 and the lysine. This extension resulted in cleavage of a C-terminal lysine (GGSGK) (SEQ ID NO: 298). Protecting cleavage of the lysine by addition of a C-terminal leucine (GGSGKL) (SEQ ID NO: 287) resulted in the LCs being 77.8% transamidated.

TABLE 3 Transamindation of a C-terminal lysine addition to the LC Z-Gln-Gly-CAD-biotin: +631 Da % Calculated Observed ΔMass conjugated Antibody 01-LC-K 23340 23343 3  0.0% 23340 23344 4   Antibody 01-LC-KL 23453 23457 4  0.0% Antibody 01-LC-LK 23453 23457 4  0.0% Antibody 01-LC-LKL 23566 23569 3  9.1% 23566 23570 4   23566 24201 635   Antibody 01-LC-GGSGK 23598 23473 −125  0.0% 23598 23474 −124 Antibody 01-LC-GGSGKL 23711 23714 3 77.8% 23711 24345 634 MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight. The masses of the reduced LCs were analyzed by ESI-MS, and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as in (Table 1).

Example 6: Analysis of Additional Acyl Donors

One utility of conjugations to an acyl acceptor on a mAb is for the manufacturing of site-specific ADCs. Conjugation of functional agents to a mAb could be achieved by one of two methods. First, a 2-step method would require mTGase conjugation of a lysine to an acyl donor synthesized with a reactive group such as BCN, DBCO, TCO, azido (N₃), alkyne, tetrazine, or maleimide. The second step involves conjugation of a functional agent to the reactive group using, for example, copper-free click chemistry or thiol-reactive chemistry. Z-Gln-Gly-N₃ is not available commercially; therefore aminopropyl-N₃ was added to the hydroxyl group of Z-Gln-Gly as detailed in the Methods section. Antibody 01 with lysine mutations in the HC or LC were incubated with Z-Gln-Gly-N3 or Z-Gln-Gly-PEG₂-BCN and mTGase as above. The samples were desalted and analyzed by LC-MS to determine addition of the substrate to the mAb. Z-Gln-Gly-N₃ was added efficiently (>75% conjugation or DAR 1.5) for most lysine substitutions (Table 4). The most permissible sites of transamidation were HC-S135K, HC-L193K, HC-D221K, HC-M252K, HC-N297K, HC-P445K, and LC-L201K. Mutants HC-T136K, HC-T223K, HC-T225K, LC-S202K, and LC-GGSGKL SEQ ID NO: 287) were all transamidated >75% with Z-Gln-Gly-CAD-biotin (FIG. 7), but not with Z-Gln-Gly-N₃. Therefore, not all acyl donors equally transamidate the same acyl acceptor site. This was also demonstrated by Z-Gln-Gly-PEG₂-BCN where no acyl acceptor site was efficiently transamidated (Table 4). Contrary to Z-Gln-Gly-N₃, the percentage of transamidation by Z-Gln-Gly-PEG₂-BCN did not vary widely among the acyl acceptor sites. For example, there was a 32% difference in transamidation of T135K and S136K by Z-Gln-Gly-N₃, but only a 4% difference with Z-Gln-Gly-PEG₂-BCN.

TABLE 4 Transamidation with various acyl donors HC Z-Gln- Z-Gln-Gly- Z-Gln-Gly- DAR Mutation Gly-N₃ PEG₂-BCN PEG₂-AuF Species T135K 89.1% 27.1% 51.4% 1 S136K 59.6% 23.1% 16.1% 1 L193K 81.5% 63.7% 23.3% 1 D221K 55.1% 31.5% 39.7% 1  8.5% 2 T223K 48.7%  0.0% ND 1 H224K 55.7% 17.3% ND 1 T225K 34.1%  0.0% ND 1 M252K 89.0% 15.6% 82.8% 1 N297K 27.6%  6.0% 46.5% 1 41.3%  0.0% 30.4% 2 23.7%  0.0%  4.5% 3 P445K 73.0% 49.6% 48.7% 1 MAbs were incubated with Z-Gln-Gly-N₃, Z-Gln-Gly-PEG₂-BCN, or Z-Gln-Gly-PEG₂-AuF and mTGase at 37° C. overnight. The masses of the reduced mAbs were analyzed by LC-MS (data not shown), and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as in (Table 1).

Interestingly, conjugation to D221K and N297K resulted in multiple conjugation sites in the HC. The D221K mutation is adjacent to Lys222, which is not typically an acyl acceptor site. Perhaps the presence of an adjacent lysine facilitates a low level of transamidation of Lys222. Of note, only the Z-Gln-Gly-N₃ substrate is conjugated at more than one site in the D221K mutant. The structure of Z-Gln-Gly-N₃ is smaller than the other substrates tested (FIG. 2), and it is likely that steric hindrence of the second conjugation site blocks its transamidation by the other substrates.

The N297K mutation removes the glycosylation site at Asn297. Aglycosylated mAbs adopt a different structure than the glycosylated forms. The N297Q mutation also results in an aglycosylated mAb and perturbs the structure in such a way that Gln295 is then transamidated by a variety of acyl acceptor substrates (Mindt, T. L. et al., 2008, Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase, Bioconjug. Chem 19:271-278; Jeger,S. et al., 2010, Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase, Angew. Chem Int Ed Engl 49:9995-9997; Dennler, P.et al., 2014, Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates, Bioconjug. Chem 25:569-578). N297K likely changes the confirmation of the CH2 region resulting in native lysines now becoming acyl acceptors sites for Z-Gln-Gly-N₃.

A second conjugation method involves a single conjugation step whereby a functional agent is synthesized with an acyl donor group. This method was tested by synthesizing a Z-Gln-Gly group onto PEG₂-Auristatin F (Z-Gln-Gly-PEG₂-AuF). The Z-Gln-Gly-PEG₂-AuF was incubated with Antibody 01 HC and LC lysine mutants and mTGase overnight at 37° C. The samples were desalted and analyzed by LC-MS to determine addition of the substrate to the mAb. All HC mutants tested demonstrated various amounts of conjugation (Table 4). The efficiency of conjugation at certain sites did not correspond with the efficiency of conjugation seen with Z-Gln-Gly-N₃. Conjugation to Z-Gln-Gly-PEG₂-AuF was low for T135K, L193K, and D221K but high for Z-Gln-Gly-N₃. L201K was the most efficient with 92% of the LCs conjugated to Z-Gln-Gly-PEG₂-AuF. N297K again demonstrated multiple conjugation sites, but the efficiency was not as high as with Z-Gln-Gly-N₃.

Example 7: Generating Multiple Acyl Acceptor Sites on a Single Antibody

A mutant mAb with one engineered acyl acceptor site yields a theoretical DAR of 2. By combining multiple engineered acyl acceptor sites, the theoretical DAR increases by 2 for each site. Increasing the drug load of an ADC results in delivery of more cytotoxic drugs into a target cell per mAb, which may allow for lower patient dosing. To determine whether multiple acyl acceptor sites could yield a mAb with a ratio of functional agent to immunoglobulin of greater than 2, mAbs were engineered to include the LC mutant L201K or S202K in combination with the CH1 mutations T135K or S136K, the CH1-CH2 mutations S136K-N297K, the CH1-CH2-CH3 mutations S136K-N297K-P445K, or the CH1-CH3 mutations T135K-L448 to yield mAbs with 4, 6, 8 or 6 acyl acceptor sites, respectively.

T135K-based samples were incubated with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C., and their masses were analyzed by LC-MS following reduction. The L201K LCs were 100% conjugated in all samples (Table 5). The S202K LC in the single T135KHC mutant had more than double the efficiency of the double T135K-L448HC mutant (69.8% versus 31.8%). T135K was 100% conjugated when combined with just the L201KLC mutation, resulting in a DAR of 4.0. In combination with the S202KLC mutation, conjugation was reduced to 88.7% with an average DAR of 3.17. Combining T135K with the L448HC mutation resulted in 100% conjugation to the mAbs. However, the DAR was heterogeneous with 77.1% containing 2 biotins per HC and 22.9% containing only 1 biotin yielding an average DAR of 3.54. When the double-HC mutations were combined with a LC mutation, conjugation efficiency to the HC dropped. The amount of DAR 1 species more than doubled and the DAR 2 species dropped 2.3- to 6.5-fold. Despite these two mAb having a potential DAR of 6, the average DAR was less than 4.

TABLE 5 Combining lysine mutations resulted in multiple acyl acceptor sites on a single mAb % HC % LC Ave conjugation conjugation DAR Z-Gln-Gly-CAD-biotin DAR 1 DAR 2 DAR 1 T135KHC/L201KLC 100.0%  0.0% 100.0% 4.00 T135KHC/S202KLC  88.7%  0.0%  69.8% 3.17 T135K-L448HC  22.9% 77.1%  0.0% 3.54 T135K-L448HC/L201KLC  56.0% 11.9% 100.0% 3.59 T135K-L448HC/S202KLC  52.8% 33.2%  31.8% 3.02 % CH1 % LC conjugation conjugation Ave DAR 1 DAR 1 DAR S136KHC/S202KLC 85.5% 72.5% 3.16 S136K-N297KHC/S202KLC 88.1% 59.7% 2.95 S136K-N297K-P445KHC/S202KLC 84.5% 56.0% 2.80 MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight. The masses of the reduced T135K mutants were analyzed by LC-MS, and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as above. The S136K mutant mAbs were digested with IdeS, reduced, and analyzed by ESI-MS. The signal from the Fc fragments was too low to analyze.

Samples containing the S136KHC mutation were incubated with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C., and their masses were analyzed by ESI-MS following IdeS digestion and reduction. The CH1 and light chain of all samples were transamidated (Table 5). S136K was 84.5% to 88.1% conjugated for all samples. S202K transamidation was higher in the single S136K mutant versus the double and triple HC mutants (72.5% versus 56% or 59.7%). The DAR for S136KHC/S202KLC was 3.16 out of 4 potential sites. The ESI-MS signal for the Fc fragments containing the N297K mutation was very low, and the conjugation efficiency could not be determined. Therefore the DARs for the double- and triple-HC mutants were at least 2.95 and 2.8.

Transamidation of multiple sites by other acyl donor substrates was also examined. The T135K-based mutants were incubated with Z-Gln-Gly-N₃, Z-Gln-Gly-PEG₂-BCN, or Z-Gln-Gly-PEG₂-AuF and mTGase overnight at 37° C. Conjugation to the LC L201K and S202K sites by each of the substrates was similar between these multiple-mutation mAbs and the single L201K and S202K mutations shown in Table 4, except no conjugation of Z-Gln-Gly-PEG₂-BCN to the LC mutations was observed (FIG. 11). The two mutants containing a single T135K mutation plus a single LC mutation demonstrated conjugation efficiency to T135K similar to the single T135K in Table 4 for all acyl donor substrates. Adding the second mutation L448 mutation resulted in mixtures of samples with DAR 1 and DAR 2 on the HC. The samples transamidated most efficiently were T135KHC/L201KLC+Z-Gln-Gly-N₃ (DAR 3.57 out of 4), T135K-L448HC+Z-Gln-Gly-N₃ (DAR 3.1 out of 4), and T135K-L448HC/L201KLC+Z-Gln-Gly-N₃ (DAR 5.04 out of 6).

Example 8: Analysis of the Lysine Substitutions in Other IgG Isotypes

The Fc of IgG1, IgG2, IgG3, and IgG4 are 89.2% identical (FIG. 10, alignment sequence), therefore it was possible that lysine insertions or substitutions at positions in IgG2, IgG3, and IgG4 analogous to IgG1 could be engineered acyl acceptor sites for mTGase. First, wild-type IgG2, IgG3, and IgG4 were analyzed to determine whether there are any native acyl acceptor sites specific to these isotypes. MAbs were incubated with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C. The samples were digested with IdeS and the masses of the Fcs were analyzed by ESI-MS. As with IgG1, there was no transamidation of wild-type IgG2, IgG3, or IgG4 (Table 6).

TABLE 6 IgG2, IgG3, and IgG4 have no acyl acceptor sites. Z-Gln-Gly-CAD-biotin: +631 Da % Glycan Calculated Observed ΔMass conjugated IgG2 G0F 25362 25232 −130 0.0% G1F 25525 25394 −131 IgG3 G0F 25396 25266 −130 0.0% G1F 25559 25428 −131 IgG4 G0F 25344 25214 −130 0.0% G1F 25507 25376 −131 MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight, followed by digestion with IdeS to generate F(ab′)₂ and Fc fragments. The masses of the IdeS-generated Fc fragments were analyzed by ESI-MS as above, and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as disclosed herein.

Lysine substitutions were made at positions analogous to IgG1 (M252, N297, and P445). Except for IgG4 that encodes for a leucine at position 445, there are no differences at these residues between the isotypes (FIG. 10). Mutant mAbs were incubated with mTGase and Z-Gln-Gly-CAD-biotin overnight at 37° C. The samples were digested with IdeS and the masses of the Fcs were analyzed by ESI-MS. The mutants N297K and M252K were efficiently transamidated with the N297K mutants yielding more than one conjugation site per HC as with IgG1 (Table 7). IgG2-N297K and IgG4-N297K contained 2 acyl acceptor sites while IgG3 contained 3. The mutant P445K was only efficiently transamidated for the IgG2 isotype. P445K transamidation was only 62.6% and 50.6 for IgG3 and IgG4, respectively.

TABLE 7 Engineered acyl acceptor sites in IgG1 Fc were also acceptor sites in IgG2, IgG3, and IgG4. ZQG-CAD-biotin = 631 Da Calculated Glycan Observed ΔMass % of total % conjugation IgG2 N297K 23803 — 24434 631 78.6% 100.0% 25059 1256 21.4% IgG3 N297K 23837 — 24468 631 28.2% 100.0% 25099 1262 40.8% 25730 1893 31.0% IgG4 N297K 23785 — 24416 631 81.6% 100.0% 25047 1262 18.4% IgG2 M252K 25231 G0F 25231 0 14.2% 85.8% 25231 G0F 25862 631 69.5% 25394 G1F 26024 630 16.3% IgG3 M252K 25265 G0F 25265 0 7.4% 92.6% 25265 G0F 25896 631 50.7% 25428 G1F 26058 630 41.9% IgG4 M252K 25213 G0F 25844 631 57.4% 100.0% 25376 G1F 26006 630 42.6% IgG2 P445K 25265 G0F 25895 630 64.7% 100.0% 25428 G1F 26058 630 35.3% IgG3 P445K 25299 G0F 25299 0 21.1% 61.6% 25462 G1F 25461 −1 17.3% 25299 G0F 25930 631 34.3% 25462 G1F 26092 630 27.4% IgG4 L445K 25231 G0F 25231 0 25.7% 50.6% 25394 G1F 25393 −1 23.7% 25231 G0F 25862 631 27.6% 25394 G1F 26024 630 23.0% MAbs were incubated with Z-Gln-Gly-CAD-biotin and mTGase at 37° C. overnight, followed by digestion with IdeS to generate F(ab′)₂ and Fc fragments. The masses of the IdeS-generated Fc fragments were analyzed by ESI-MS as above, and the percent conjugation to Z-Gln-Gly-CAD-biotin (Δmass = 631 Da) was determined as disclosed herein.

Example 9: Transamidation Sites in the Fab′ Region

The hinge of IgG is a flexible linker between CH1 and CH2, and it is therefore possible that this flexibility would allow transamidation of acyl acceptors in this region. Zhang, et al., 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography, Sci Rep, (2015) 5:9803. Indeed, upper hinge mutants D221K, T223K, H224K, and T225K are efficiently transamidated; however, there was no transamidation of middle or lower hinge mutants. This lack of transamidation may be due to structural constraints of the interchain disulfide bonds and proximity to CH2, respectively. To determine whether these residues can be transamidated when relieved of any structural constraints, lysine mutations were made in the context of a Fab′ containing the entire hinge region with Cys226 and Cys229 mutated to alanine (DTHTAPPAPAPELL) (SEQ ID NO: 299).

The transamination of mutant Fab's was determined by ESI-MS as above using Z-Gln-Gly-CAD-biotin as the acyl donor. The masses of the HC portion of the Fab's were as expected except for the mutant L235K. This mutation results in a C-terminal lysine residue that was cleaved, likely due to carboxypeptidase B as with full length IgG. Harris et al., Structural characterization of a recombinant CD4-IgG hybrid molecule, Eur J Biochem, (1990) 194:611-620; Harris, Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture, J Chromatogr.A, (1995) 705:129-134; Dick, Jr. et al., C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes, Biotechnol. Bioeng., (2008) 100:1132-1143. While no transamidation of full-length IgG was seen, a minor amount of transamidation (4.7%) was seen in the wild-type Fab′, possibly at Lys222 (FIG. 9). Scanning lysine mutagenesis of the Fab′ revealed several acyl acceptor sites at positions D221, T223, H224, T225, P230, and E233. These Fab's were transamidated with 1 or 2 biotins, with one on the engineered lysine and the second on a native lysine.

As secondary structure plays a large part in determining whether a lysine is transamidated, and the secondary structure of the hinge between the Fab′ mutants is unlikely to change significantly, it was unexpected that the native Lys222 and mutants C226K, P227K, C229K, A231K, P232K, L234K, and L235K were not transamidated. The primary sequences surrounding the lysine mutations were analyzed. It was previously demonstrated that a residue C-terminal (+1) to an acyl acceptor affect transamidation; specifically a +1 acidic or proline residue results in little to no transamidation see, e.g., U.S. Provisional No. 62/269,138 filed on Dec. 18, 2015, and PCT/US2016/067165 filed on Dec. 16, 2016, the entire contents of each of which are expressly incorporated herein by reference. The mutants C226K, P227K, C229K, and A231K all have a +1 proline and P232K has a +1 glutamate. In addition, Lys222 and L234K have a −1 acidic residue. It is possible that any acidic residue flanking an acyl acceptor inhibits transamidation. Therefore, alanine mutations were made to mutate either the −1 or +1 acidic residue or +1 proline. Indeed, mutating the acidic and proline residues resulted in efficient transamidation of the lysine substitutions (Table 8). The D221A mutation increased transamidation from 5% to 66%. Similarly, mutating the −1 or +1 acid residues in the P232K,E233A and E233A,L234K Fab's resulted in increased transamidation from 46% to 93% and 62% to 86%, respectively. Mutation of a +1 proline had similar results for lysine mutants C226K (52% to 71%), P227K (57% to 81%), C229K (33% to 91%), and A231K (22% to 93%).

The effect of the hinge length on transamidation of mutants T223K, H224K, T225K, P228K, P230K, and E233K was analyzed by deleting all but the adjacent+1 residue. Removing the C-terminal hinge residues had no negative effect on transamidation of the engineered acyl acceptor site for mutants T223K, H224K, and T225K, and increased transamidation of mutants P228K, P230K, and E233K (Table 8).

TABLE 8 (SEQ ID NOS: 300-326) Hinge lysine mutations in a Fab′, but not a mAb, were acyl acceptors. % conjugation +1 biotin +2 biotins Total M9-Fab DKTHTAPPAPAPELL  4.7%  4.7% D221A AKTHTAPPAPAPELL 65.9% 65.9% D221K KKTHTAPPAPAPELL 89.6% 89.6% T223K DKKHTAPPAPAPELL 51.1% 34.2% 85.3% T223K-H DKKH 56.6% 25.9% 82.5% H224K DKTKTAPPAPAPELL 87.7%  2.6% 90.2% H224K-T DKTKT 83.6%  5.4% 89.0% T225K DKTHKAPPAPAPELL 69.6% 12.2% 81.8% T225K-A DKTHKA 62.6% 14.2% 76.8% C226K DKTHTKPPAPAPELL 52.2% 52.2% C226K, P228A DKTHTKAPAPAPELL 60.1% 11.2% 71.3% P227K DKTHTAKPAPAPELL 56.8% 56.8% P227K, P228A DKTHTAKAAPAPELL 62.6% 17.9% 80.5% P228K DKTHTAPKAPAPELL 16.5% 16.5% P228K-A DKTHTAPKA 65.8% 28.7% 94.4% C229K DKTHTAPPKPAPELL 32.5% 32.5% C229K, P230A DKTHTAPPKAAPELL 77.3% 13.3% 90.6% P230K DKTHTAPPAKAPELL 63.9% 14.8% 78.7% P230K-A DKTHTAPPAKA 75.7% 17.1% 92.8% A231K DKTHTAPPAPKPELL 22.0% 22.0% A231K, P232A DKTHTAPPAPKAELL 79.3% 13.6% 92.9% P232K DKTHTAPPAPAKELL 40.6%  5.5% 46.2% P232K, E233A DKTHTAPPAPAKALL 85.0%  7.7% 92.6% E233K DKTHTAPPAPAPKLL 51.5% 51.5% E233K-L DKTHTAPPAPAPKL 82.4%  9.0% 91.4% L234K DKTHTAPPAPAPEKL 53.2%  8.5% 61.7% E233A, L234K DKTHTAPPAPAPAKL 75.4% 10.9% 86.3% L235K DKTHTAPPAPAPELK  7.8%  7.8% Purified mutant hinge mutant Fab's were screened for transamidation by incubating mTGase with Z-Gln-Gly-CAD-biotin overnight at 37° C. The masses of the HC were analyzed by ESI-MS and the percentage of conjugation was determined.

Example 10: Generation of Dimeric Antibody Molecules

In addition to mTGase mediating transamidation between a small molecule acyl donor and a large molecule acyl acceptor, the transamidation of mutant Fabs with a mAb containing an acyl donor was analyzed. The mAb mutant N297Q contains two acyl donor sites at positions 295 and 297. Jeger et al., Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase, Angew. Chem Int Ed Engl, (2010) 49:9995-9997. Fab′ mutants D221K, H224K, T225K, P228K, P230K, and E233K were incubated with N297Q and mTGase overnight at 37° C. The samples were reduced and analyzed on by SDS-PAGE. Unconjugated mAb and Fab′ will yield bands at 23 kDa (LC), 25 kDa (Fab-Vh-CH1), 38 kDa (mTGase), and 49 kDa (HC). A single mAb-Fab′ conjugation will result in a band at 73 kDa and conjugation of two Fab's to a mAb will result in a band at 98 kDa. Indeed, all samples contain bands at both ˜75 and ˜100 kDa (FIG. 11).

Conclusion to Examples

Given the number of native lysines in exposed loops and turns within an antibody, it was unexpected to find no suitable acyl acceptors on any antibody tested. These results are in direct contrast to the prior thinking in the field, including those reported by Mindt et al., 2008), who allegedly demonstrated conjugation of an average of 0.3 molecules of a fluorescent dye to a natural lysine residue on the antibody chCE7. Mindt also allegedly reported a low level (0.1 molecules/antibody) of transamidation of an acyl acceptor substrate to the same antibody, chCE7. However, numerous later publications could not replicate the findings of Mindt (see Strop et al., 2013; Jeger et al., 2010; and Siegmund et al., 2015). Specifically, Jeger noted that “no modification of native chCE7 or RTX was observed with any of the substrates” (see Jeger et al., 2010). Therefore, the findings disclosed in Mindt were likely either due to background fluorescence of the assay or specific to the acyl acceptor or donor being located in the variable regions of chCE7.

While no acyl acceptors were found on wild-type monoclonal antibodies, single amino acid lysine substitutions surprisingly revealed several positions throughout an antibody that can be engineered to be acyl acceptors. At least one site was identified in each of the constant region domains of the HC and LCs (FIG. 12). Beyond their position in solvent-exposed loops or turns, there was no clear consensus sequence surrounding the acyl acceptor sites. Examples of sequences of the sites that were transamidated greater than 70% were KSK₁₃₅SG (SEQ ID NO: 327), STK₁₃₆GG (SEQ ID NO: 328), SSK₁₉₃GT (SEQ ID NO: 329), TLK₂₅₂IS (SEQ ID NO: 330), QYK₂₉₇ST (SEQ ID NO: 331), LSK₄₄₅G* (SEQ ID NO: 332), QGK₂₀₁SS (SEQ ID NO: 333), GLK₂₀₂SP (SEQ ID NO: 334), PTK₂₁₃CS (SEQ ID NO: 335). Similar sequences are found surrounding lysines that were not transamidated, such as in CH1 (SSK₁₃₃ST) (SEQ ID NO: 336), CH3 (LTK₃₆₀NQ) (SEQ ID NO: 337), and kappa (QLK₁₂₆SG (SEQ ID NO: 338), LSK₁₈₃AD (SEQ ID NO: 339)).

An acidic or proline residue flanking a lysine did hinder the efficiency of transamidation, as demonstrated by mutations in the hinge of Fabs. In addition to mutating a residue to a lysine, acyl acceptor site can be engineered by mutating residues flanking an existing lysine. Based on the transamidation of Lys222 in the Fab mutant D221A, mutation of other proline or acidic residues flanking native lysine throughout the mAb is possible. For instance, IgG1 lysines PK₂₄₆P, PK₂₄₈D, TK₂₉₀P, GK₃₁₇E, and DK₄₁₄S, kappa lysines SK₁₆₉D and EK₁₈₈H, and lambda lysine EK₂₀₇T are flanked by acidic and proline residues, and mutation to something other than an acidic or proline residue may create an acyl acceptor site at that particular lysine. Further, it may be possible that some sites were missed in the lysine scanning mutagenesis due to the mutant lysine being adjacent to an acidic or proline residue, as was the case for several Fab hinge mutants.

The structure surrounding the residue was also shown to influence transamidation. By relieving the core and lower hinge region of any structure contributed by CH2 and/or the interchain disulfide bonds through the generation of Fab fragments lysines that were not transamidated in the context of a mAb were transamidated in a Fab. In the context of a full-length mAb with interchain disulfide bonds at cysteines 226 and 229, P230K and E233K were not transamidated. However, in the context of a Fab fragment and no interchain disulfide bonds these residues were transamidated. A disulfide bond flanking an acyl acceptor site in amongst itself does not hinder transamidation, as the +1 position in the lambda mutant E213K is an interchain disulfide bond, and the mutant was transamidated. Therefore, either changing the three-dimensional structure of the hinge region by removing the interchain disulfide bonds and/or removing possible steric constraints of the nearby CH2 domain allowed transamidation of lysines at these sites.

MTGase has previously been investigated as a means to transamidate glutamines by engineering mAbs by one of two methods. Josten, et al., Use of microbial transglutaminase for the enzymatic biotinylation of antibodies, J Immunol.Methods, (2000) 240:47-54; Mindt, et al., Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase, Bioconjug.Chem, (2008) 19:271-278; Jeger, et al., Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase, Angew.Chem Int Ed Engl, (2010) 49:9995-9997; Strop, et al., Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates, Chem Biol, (2013) 20:161-167. While effective in conjugating amine-based substrates on mAbs, both methods have disadvantages. The first approach requires deglycosylation of the mAb either enzymatically or by mutagenesis. Mindt, et al., Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase, Bioconjug.Chem, (2008) 19:271-278; Jeger, et al., Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase, Angew.Chem Int Ed Engl, (2010) 49:9995-9997. Aglycosylated mAbs undergo a conformational change that decreases the thermal stability, increases protease susceptibility, and increases aggregation rates. Mimura, et al., The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms, Mol Immunol., (2000) 37:697-706; Kwon, et al., Effect of glycosylation on the stability of alphal-antitryp sin toward urea denaturation and thermal deactivation, Biochim Biophys.Acta, (1997) 1335:265-272; Wang, et al., pH dependent effect of glycosylation on protein stability, Eur J Pharm Sci, (2008) 33:120-127; Yamaguchi, et al., Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy, Biochim Biophys.Acta, (2006) 1760:693-700; Arnold, et al., The impact of glycosylation on the biological function and structure of human immunoglobulins, Annu Rev Immunol., (2007) 25:21-50; Zheng, et al., The impact of glycosylation on monoclonal antibody conformation and stability, MAbs., (2011) 3:568-576. The substitution N297K resulted in a site of transamidation, and utilizing this site also has the same disadvantage as the N297Q mutation.

The second approach involves engineering a 4-amino acid LLQG glutamine tag at either termini or within a solvent exposed region of the mAb. Strop, et al., Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates, Chem Biol, (2013) 20:161-167. The addition of 4 amino acids increases the likelihood of an immunogenic response in a patient which would reduce the efficacy of the ADC. In contrast, the modifications described herein utilize single amino acid substitutions or single amino acid, e.g., lysine, insertions as conjugation sites, thereby reducing the chance of eliciting an immune response in patients. Furthermore, multiple sites throughout the IgG, kappa, and lambda constant domains can be used if it is found that one site confers an undesirable property on the mAb such as increased aggregation or immunogenicity.

In summary, acyl acceptor sites can be engineeried in an antibody, or antigen binding fragment, by either insertion of a lysine between two native residues or by a lysine substitution at various positions throughout IgG1-4, kappa, or lambda. The optimal context for an acyl acceptor site is not only position-dependent, but also requires no acidic residue at the −1 or +1 position or no proline at the +1 position. Microbial transglutaminase conjugation technology can utilize these engineered sites to conjugate a variety of acyl donor-containing functional agents for making ADCs, bispecific antibodies, immunotoxins, or other mAb-protein complexes.

Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

INFORMAL SEQUENCE LISTING SEQ ID NO Antibody/Domain Sequence 1 4F3F-VH QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVK QSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSS TAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC 2 4F3F-VK DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKS GTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEA EDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 3 4HK0-VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV RQAPGQGLEWMGWINPNSGGTNYAQKFQGWVTMTRD TSISTAYMELSRLRSDDTAVYYCARGGLEPRSVDYYYY GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKHHHHHH 4 4HK0-VK QSVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKP GQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEA GDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPS VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSS PVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS 5 1FC1 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNST YRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 6 Antibody 02HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 7 Antibody 02LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 8 Antibody 03HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGFFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 9 Antibody 03LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10 Antibody 01HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 11 Antibody 01LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 12 Antibody 04HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 13 Antibody 04LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 14 Antibody 05HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 15 Antibody 05LC GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 16 Antibody 06HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 17 Antibody 06LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 18 human gamma 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 19 human kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 human lambda GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHKSYSCQVTHEGSTVEKTVAPTECS 21 Antibody 01HC- KSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A118K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 22 Antibody 01HC- AKTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV S119K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 23 Antibody 01HC- ASKKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV T120K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 24 Antibody 01HC- ASTKKPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G122K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 25 Antibody 01HC- ASTKGPSVFPLAPKSKSTSGGTAALGCLVKDYFPEPVTV S131K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 26 Antibody 01HC- ASTKGPSVFPLAPSKKSTSGGTAALGCLVKDYFPEPVTV S132K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 27 Antibody 01HC- ASTKGPSVFPLAPSSKKTSGGTAALGCLVKDYFPEPVTV S134K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 28 Antibody 01HC- ASTKGPSVFPLAPSSKSKSGGTAALGCLVKDYFPEPVTV T135K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 29 Antibody 01HC- ASTKGPSVFPLAPSSKSTKGGTAALGCLVKDYFPEPVTV S136K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 30 Antibody 01HC- ASTKGPSVFPLAPSSKSTSKGTAALGCLVKDYFPEPVTVS G137K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 31 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGKTAALGCLVKDYFPEPVTVS G138K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 32 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGKAALGCLVKDYFPEPVTV Y319K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 33 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPKPVTV E152K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 34 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEKVTV P153K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 35 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S160K WNKGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 36 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A162K WNSGKLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 37 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L163K WNSGAKTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 38 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T164K WNSGALKSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 39 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S165K WNSGALTKGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 40 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G166K WNSGALTSKVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 41 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS V167K WNSGALTSGKHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 42 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S176K WNSGALTSGVHTFPAVLQKSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 43 Antibody 01HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S177K WNSGALTSGVHTFPAVLQSKGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 44 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G178K WNSGALTSGVHTFPAVLQSSKLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 45 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L179K WNSGALTSGVHTFPAVLQSSGKYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 46 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P189K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVKSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 47 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S190K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPKSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 48 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S191K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSKSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 49 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S192K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 50 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L193K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSKGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 51 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G194K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLKTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 52 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T195K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGKQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 53 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q196K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 54 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T197K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ KYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 55 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P206K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKKSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 56 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S207K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPKNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 57 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E216K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVKPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 58 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P217K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEKKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 59 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S219K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKKCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 60 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D221K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCKKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 61 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T223K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKKHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 62 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H224K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTKTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 63 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T225K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHKCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 64 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C226K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTKPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 65 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P227K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCKPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 66 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P228K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPKCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 67 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C229K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPKPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 68 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P230K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCKAPELL GGPSVFLFPPKPKDTLMISRTPEVCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 69 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A231K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPKPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 70 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P232K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAKELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 71 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E233K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPKLL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 72 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L234K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEKL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 73 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L235K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELK GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 74 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G236K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL KGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 75 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G237K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GKPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 76 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P247K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKKKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 77 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS M252K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLKISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 78 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS I253K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMKSRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 79 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S254K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMIKRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 80 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS R255K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISKTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 81 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T256K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRKPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 82 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D265K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVKVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 83 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S267K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 84 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H268K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSKEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 85 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E269K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHKDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 86 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D270K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEKPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 87 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P271K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDKEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 88 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E272K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPKVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 89 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D280K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVKGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 90 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G281K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDKVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 91 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS V282K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGKEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 92 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E283K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVKVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 93 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS V284K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEKHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 94 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H285K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVKNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 95 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N286K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHKAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 96 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A287K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNKKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 97 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T289K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKKKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 98 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q295K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEKYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 99 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Y296K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQKNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 100 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N297K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYKSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 101 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S298K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNKTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 102 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L309K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVKHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 103 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H310K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLKQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 104 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q311K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHKD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 105 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D312K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQK WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 106 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L314K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WKNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 107 Antibody 01HC- STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N315K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLKGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 108 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G316K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNKKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 109 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E318K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKKYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 110 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A327K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKKLPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 111 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P329K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALKAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 112 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A330K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPKPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 113 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P331K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAKIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 114 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S337K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTIKKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 115 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A339K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKKKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 116 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G341K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKKQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 117 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q342K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGKPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 118 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P343K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQKREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 119 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS R344K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPKEPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 120 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E345K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPRKPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 121 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS R355K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSKDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 122 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D356K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 123 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L358K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDEKTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 124 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T359K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELKKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 125 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N361K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKKQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 126 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q362K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNKVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 127 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S375K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPKDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 128 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D376K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSKIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 129 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E382K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWKSNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 130 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N384K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESKGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 131 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G385K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNKQPENN YKTTPPVLDSDGSFFLYKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 132 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q386K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGKPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 133 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS I387K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQKENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 134 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N389K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEKN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 135 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N390K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENK YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 136 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L398K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVKDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 137 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S400K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDKDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 138 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D401K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSKGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 139 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G402K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDKSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 140 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D413K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVKKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 141 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S415K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKKRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 142 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS R416K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSKWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 143 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q418K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWKQGNVFSCSVM HEALHNHYTQKSLSLSPGK 144 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Q419K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQKGNVFSCSVM HEALHNHYTQKSLSLSPGK 145 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G420K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQKNVFSCSVM HEALHNHYTQKSLSLSPGK 146 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N421K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGKVFSCSVM HEALHNHYTQKSLSLSPGK 147 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS V422K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNKFSCSVM HEALHNHYTQKSLSLSPGK 148 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A431K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEKLHNHYTQKSLSLSPGK 149 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H433K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALKNHYTQKSLSLSPGK 150 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N434K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHKHYTQKSLSLSPGK 151 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H435K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNKYTQKSLSLSPGK 152 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Y436K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHKTQKSLSLSPGK 153 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S442K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLKLSPGK 154 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L443K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSKSPGK 155 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S444K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLKPGK 156 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P445K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSKGK 157 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS G446K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPKK 158 Antibody 01LC- KTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV R108K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 159 Antibody 01LC- RKVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV T109K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 160 Antibody 01LC- RTKAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV V110K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 161 Antibody 01LC- RTVAKPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV A112K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 Antibody 01LC- RTVAAPSVFIFPPSKEQLKSGTASVVCLLNNFYPREAKV D122K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 163 Antibody 01LC- RTVAAPSVFIFPPSDKQLKSGTASVVCLLNNFYPREAKV E123K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 164 Antibody 01LC- RTVAAPSVFIFPPSDEQLKKGTASVVCLLNNFYPREAKV S127K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 165 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSKTASVVCLLNNFYPREAKV G128K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 166 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGKASVVCLLNNFYPREAKV T129K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 167 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPKEAKV R142K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 168 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRKAKV E143K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 169 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV D151K QWKVKNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 170 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV N152K QWKVDKALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 171 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV A153K QWKVDNKLQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 172 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV L154K QWKVDNAKQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 173 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV Q155K QWKVDNALKSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 174 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV S156K QWKVDNALQKGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 175 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV G157K QWKVDNALQSKNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 176 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV E165K QWKVDNALQSGNSQESVTKQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 177 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV D167K QWKVDNALQSGNSQESVTEQKSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 178 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV S168K QWKVDNALQSGNSQESVTEQDKKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 179 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV D170K QWKVDNALQSGNSQESVTEQDSKKSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 180 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV S182K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLKKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 181 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV A184K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKK DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 182 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV E187K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYKKHKVYACEVTHQGLSSPVTKSFNRGEC 183 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV H189K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKKKVYACEVTHQGLSSPVTKSFNRGEC 184 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV V191K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKKYACEVTHQGLSSPVTKSFNRGEC 185 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV Q199K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHKGLSSPVTKSFNRGEC 186 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV G200K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQKLSSPVTKSFNRGEC 187 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV L201K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGKSSPVTKSFNRGEC 188 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV S202K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLKSPVTKSFNRGEC 189 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV S203K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSKPVTKSFNRGEC 190 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV P204K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSKVTKSFNRGEC 191 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV N210K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFKRGEC 192 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV R211K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNKGEC 193 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV G212K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRKEC 194 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV E213K QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGKC 195 Antibody 05LC- KQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV G110K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 196 Antibody 05LC- GKPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV Q111K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 197 Antibody 05LC- GQKKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT P112K VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS 198 Antibody 05LC- GQPKAKPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV A115K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 199 Antibody 05LC- GQPKAAPSVTLFPPSKEELQANKATLVCLISDFYPGAVT S125K VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS 200 Antibody 05LC- GQPKAAPSVTLFPPSSKELQANKATLVCLISDFYPGAVT E126K VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS 201 Antibody 05LC- GQPKAAPSVTLFPPSSEEKQANKATLVCLISDFYPGAVT L128K VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE QWKSHRSYSCQVTHEGSTVEKTVAPTECS 202 Antibody 05LC- GQPKAAPSVTLFPPSSEELKANKATLVCLISDFYPGAVTV Q129K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 203 Antibody 05LC- GQPKAAPSVTLFPPSSEELQKNKATLVCLISDFYPGAVTV A130K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 204 Antibody 05LC- GQPKAAPSVTLFPPSSEELQAKKATLVCLISDFYPGAVTV N131K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 205 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPKAVTV G145K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 206 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGKVTV A146K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 207 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAKTV V147K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 208 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S155K AWKADKSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 209 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S156K AWKADSKPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 210 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV P157K AWKADSSKVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 211 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV A160K AWKADSSPVKKGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 212 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV G161K AWKADSSPVKAKVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 213 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S171K AWKADSSPVKAGVETTTPSKQKNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 214 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV N172K AWKADSSPVKAGVETTTPSKQSKNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 215 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV N173K AWKADSSPVKAGVETTTPSKQSNKKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 216 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV T184K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLKPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 217 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV E186K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPKQ WKSHRSYSCQVTHEGSTVEKTVAPTECS 218 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV Q187K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEK WKSHRSYSCQVTHEGSTVEKTVAPTECS 219 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S190K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKKHRSYSCQVTHEGSTVEKTVAPTECS 220 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV H191K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSKRSYSCQVTHEGSTVEKTVAPTECS 221 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV E201K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHKGSTVEKTVAPTECS 222 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV G202K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEKSTVEKTVAPTECS 223 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S203K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGKTVEKTVAPTECS 224 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV P211K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAKTECS 225 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV T212K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPKECS 226 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV E213K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTKCS 227 Antibody 05LC- GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV S215K AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECK 228 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S191.K.S192 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 229 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS S192.K.L193 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSKLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 230 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L193.K.G194 WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLKGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 231 Antibody 01-LC-K RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECK 232 Antibody 01-LC-KL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECKL 233 Antibody 01-LC-LK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECLK 234 Antibody 01LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV LKL QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECLKL 235 Antibody 01-LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV GGSGK QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGK 236 Antibody 01-LC- RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV GGSGKL QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGKL 237 Antibody 01HC- ASTKGPSVFPLAPSSKSKSGGTAALGCLVKDYFPEPVTV T135K-L448HC SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKL 238 Antibody 01HC- ASTKGPSVFPLAPSSKSTKGGTAALGCLVKDYFPEPVTV S136K-N297K SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYKSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 239 Antibody 01HC- ASTKGPSVFPLAPSSKSTKGGTAALGCLVKDYFPEPVTV S136K-N297K- SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT P445K QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYKSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSKGK 240 hu IgG2 AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD WLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 241 hu IgG3 GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENN YNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVM HEALHNRFTQKSLSLSPGK 242 hu IgG4 GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK 243 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG2HC WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 244 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG2HC-M252K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLKISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 245 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG2HC-N297K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFKSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 246 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG2HC-P445K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQ TYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSKGK 247 Antibody ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVS 01IgG3HC WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPK SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSG QPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK 248 Antibody ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVS 01IgG3HC-M252K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPK SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PAPELLGGPSVFLFPPKPKDTLKISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSG QPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK 249 Antibody ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVS 01IgG3HC-N297K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPK SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYKSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSG QPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK 250 Antibody ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVS 01IgG3HC-P445K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPK SCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSG QPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSKGK 251 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG4HC WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 252 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG4HC-M252K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGP SVFLFPPKPKDTLKISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 253 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG4HC-N297K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFKSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 254 Antibody ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS 01IgG4HC-L445K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSKGK 255 Antibody 01FabHC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPEL L 256 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D221A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCAKTHTAPPAPAPEL L 257 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS D221K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNIIKPSNTKVDKKVEMSCKKTHTAPPAPAPEL L 258 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T223K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKKHTAPPAPAPEL L 259 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T223K-H WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKKH 260 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H224K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTKTAPPAPAPEL L 261 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS H224K-T WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTKT 262 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T225K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHKAPPAPAPEL L 263 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS T225K-A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHKA 264 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C226K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTKPPAPAPEL L 265 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C226K, P228A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTKAPAPAPEL L 266 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P227K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAKPAPAPEL L 267 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P227K, P228A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAKAAPAPE LL 268 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P228K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPKAPAPEL L 269 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P228K-A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPKA 270 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C229K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPKPAPEL L 271 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS C229K, P230A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPKAAPEL L 272 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P230K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAKAPEL L 273 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P230K-A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAKA 274 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A231K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPKPEL L 275 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS A231K, P232A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPKAEL L 276 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P232K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAKEL L 277 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS P232K, E233A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAKA LL 278 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E233K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPKL L 279 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E233K-L WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPKL 280 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L234K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPEK L 281 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS E233A, L234K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPA KL 282 Antibody 01FabHC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS L235K WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSKSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPPAPAPEL K 283 Antibody 01HC- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS N297Q WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 284 Signal sequence MGWSCIILFLVATATGVHS 

1. A method for generating a conjugated immunoglobulin, the method comprising: contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and a functional agent comprising an acyl donor substrate, a) wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, b) wherein the acyl donor substrate comprises a glutamine residue, and c) wherein the functional agent is a therapeutic agent or a diagnostic agent, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate on the functional agent, thereby generating the conjugated immunoglobulin.
 2. A method for generating a conjugated immunoglobulin, the method comprising: i) contacting an immunoglobulin, or antigen-binding portion thereof, with a microbial transglutaminase and an acyl donor substrate, a) wherein the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, and b) wherein the acyl donor substrate comprises a glutamine residue and a reactive group, wherein the microbial transglutaminase conjugates the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate, and ii) conjugating a functional agent to the reactive group of the acyl donor substrate, wherein the functional agent is a therapeutic agent or a diagnostic agent, thereby generating the conjugated immunoglobulin.
 3. The method of claim 2, wherein the reactive group of the acyl donor substrate is conjugated to the functional agent by click chemistry.
 4. The method of claim 1, wherein the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Threonine 135 (T135K), Serine 136 (S136K), Leucine 193 (L193K), Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Methionine 252 (M252K), Asparagine 297 (N297K), or Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, Leucine 201 (L201K) or Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, or Glutamic acid 213 (E213K) on a lambda light chain of the immunoglobulin, or antigen-binding portion thereof.
 5. The method of claim 4, wherein the heavy chain further comprises an amino acid residue which has been added to its C-terminus at position 448, and wherein said amino acid residue is not proline or an acidic amino acid residue.
 6. The method of claim 5, wherein the amino acid residue which has been added to the C-terminus at position 448 is leucine.
 7. The method of claim 1, wherein the lysine residue insertion is a lysine residue which has been inserted between Serine 191 and Serine 192 or between Serine 192 and Leucine 193 on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.
 8. The method of claim 1, wherein the immunoglobulin, or antigen-binding portion thereof, is a fragment-antigen binding (Fab), and wherein the natural amino acid residue which has been mutated to a lysine residue is selected from the group consisting of: Aspartic acid 221 (D221K), Threonine 223 (T223K), Histidine 224 (H224K), Threonine 225 (T225K), Proline 228 (P228K), Proline 230 (P230K), and Glutamic acid 233 (E233K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof. 9.-10. (canceled)
 11. The method of claim 1, wherein the immunoglobulin, or antigen-binding portion thereof, further comprises a second engineered lysine residue, wherein the second engineered lysine residue is a second lysine residue insertion or a second natural amino acid residue which has been mutated to a second lysine residue, and wherein the microbial transglutaminase conjugates the second engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate.
 12. The method of claim 11, wherein the natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.
 13. The method of claim 11, wherein the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Leucine 201 (L201K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.
 14. The method of claim 11, wherein the natural amino acid residue which has been mutated to the engineered lysine residue is Threonine 135 (T135K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the second natural amino acid residue which has been mutated to the second engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.
 15. The method of claim 13, wherein the heavy chain further comprises an amino acid residue which has been added to its C-terminus at position 448, and wherein said amino acid residue is not proline or an acidic amino acid residue.
 16. The method of claim 15, wherein the amino acid residue which has been added to the C-terminus at position 448 is leucine.
 17. The method of claim 11, wherein the immunoglobulin, or antigen-binding portion thereof, further comprises a third engineered lysine residue, wherein the third engineered lysine residue is a third lysine residue insertion or a third natural amino acid residue which has been mutated to a third lysine residue, and wherein the microbial transglutaminase conjugates the third engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate.
 18. The method of claim 17, wherein the natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second engineered lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, and the third natural amino acid residue which has been mutated to the third engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof.
 19. The method of claim 17, wherein the immunoglobulin, or antigen-binding portion thereof, further comprises a fourth engineered lysine residue, wherein the fourth engineered lysine residue is a fourth lysine residue insertion or a fourth natural amino acid residue which has been mutated to a lysine residue, and wherein the microbial transglutaminase conjugates the fourth engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, to the glutamine residue of the acyl donor substrate.
 20. The method of claim 19, wherein the natural amino acid residue which has been mutated to the engineered lysine residue is Serine 136 (S136K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the second natural amino acid residue which has been mutated to the second engineered lysine residue is Asparagine 297 (N297K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof, the third natural amino acid residue which has been mutated to the third engineered lysine residue is Serine 202 (S202K) on a kappa light chain of the immunoglobulin, or antigen-binding portion thereof, and the fourth natural amino acid residue which has been mutated to the fourth engineered lysine residue is Proline 445 (P445K) on a heavy chain of the immunoglobulin, or antigen-binding portion thereof.
 21. The method of claim 1, wherein the amino acid residue after the engineered lysine residue is not proline or an acidic amino acid residue.
 22. The method of claim 1, wherein the amino acid residue before the engineered lysine residue is not an acidic amino acid residue.
 23. The method of claim 1, wherein the amino acid residue before the engineered lysine residue is a non-acidic amino acid residue insertion, or a natural acidic amino acid residue which has been mutated to a non-acidic amino acid residue.
 24. The method of claim 1, wherein the amino acid residue after the engineered lysine residue is an amino acid residue insertion, wherein the amino acid residue insertion is a non-acidic amino acid residue insertion and a non-proline residue insertion, or a natural acidic amino acid residue or a natural proline residue, which has been mutated to a non-acidic amino acid residue and a non-proline residue.
 25. The method of claim 23, wherein the non-acidic amino acid residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.
 26. The method of claim 23, wherein the non-acidic amino acid residue insertion is an lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan insertion.
 27. The method of claim 24, wherein the non-acidic amino acid and non-proline residue is lysine, arginine, histidine, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan.
 28. The method of claim 23, wherein the natural acidic amino acid residue is aspartic acid or glutamic acid.
 29. The method of claim 1, wherein the immunoglobulin, or antigen-binding portion thereof, comprises a heavy chain which further comprises at least one amino acid residue which has been added to its C-terminus at position 448, and wherein said at least one amino acid residue is not proline or an acidic amino acid residue.
 30. The method of claim 29, wherein the at least one amino acid residue which has been added to the C-terminus at position 448 is leucine.
 31. The method of claim 1, wherein the immunoglobulin, or antigen-binding portion thereof, comprises a light chain which comprises an insertion of one to four additional amino acids after cysteine 214, wherein the lysine residue insertion is a lysine residue which has been inserted after the one to four additional amino acids, and wherein a leucine residue has been inserted after the lysine residue.
 32. The method of claim 31, wherein the insertion of one to four additional amino acids after cysteine 214, the lysine residue which has been inserted after the one to four additional amino acids, and the leucine residue which has been inserted after the lysine residue comprises a sequence selected from the group consisting of: GKL, GGKL, GGSKL, and GGSGKL.
 33. The method of claim 1, wherein the functional agent comprising the acyl donor substrate is according to one Formulae (I) or (II): (Z)_(m)-Gln-(L)_(n)-(Y)  (I) (Y)-(L)_(n)-Gln-(Z)_(m)  (II) wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and Y is a functional agent.
 34. The method of claim 33, wherein the functional agent comprising the acyl donor substrate is according to formula (I), and wherein Z is a CBZ group; wherein each L is independently a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2, 3, 4 or
 5. 35. The method of claim 33, wherein the functional agent comprising the acyl donor substrate is according to formula (I), wherein Z is a CBZ group, and L is an amino acid.
 36. The method of claim 35, wherein L is Gly; m is 1; and n is
 0. 37. The method of claim 35, wherein L is Gly; m is 1; and n is
 1. 38. The method of claim 33, wherein the functional agent comprising the acyl donor substrate is according to formula (II), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is Gly.
 39. The method of claim 33, wherein the functional agent Y is auristatin F.
 40. The method of claim 2, wherein the acyl donor substrate is according to one Formulae (III) or (IV): (Z)_(m)-Gln-(L)_(n)-(X)  (III) (X)-(L)_(n)-Gln-(Z)_(m)  (IV) wherein Z is a carboxylbenzyloxy (CBZ) group or an amino acid residue; Gln is a glutamine amino acid residue; each L is independently a straight or branched linker from 1 to 20 carbon atoms, wherein one or more of the carbon atoms may be optionally and independently replaced with a nitrogen, oxygen or sulfur atom, and wherein each carbon and nitrogen atom may be optionally substituted; or each L is optionally and independently an amino acid residue; m is an integer from 0 to 5; n is an integer from 0 to 5; and X is a reactive group.
 41. The method of claim 40, wherein the acyl donor substrate is according to formula (III), and wherein Z is a CBZ group; wherein each L is independently a polyethylene glycol moiety (PEG) (—O((CH₂)₂)—), ethyl amine (—NH((CH₂)₂)—) or propyl amine (—NH((CH₂)₃)—); and wherein n is 0, 1, 2, 3, 4, or
 5. 42. The method of claim 40, wherein the acyl donor substrate is according to formula (III), wherein Z is a CBZ group, and wherein one or more L is an amino acid.
 43. The method of claim 42, wherein L is Gly; m is 1; and n is
 1. 44. The method of claim 42, wherein m is 1; and n is
 0. 45. The method of claim 40, wherein the acyl donor substrate is according to formula (IV), wherein Z is a CBZ group; m is 1; n is 1, 2 or 3; and at least one L is Gly.
 46. The method of claim 40, wherein X is a reactive group selected from the group consisting of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN),

(dibenzocyclooctyne; DBCO), trans-cyclooctene (TCO), azido (N₃), alkyne, tetrazine methylcyclopropene, norbornene, hydrazide/hydrazine, and aldehyde.
 47. The method of claim 1, wherein the therapeutic agent is an antibody or antigen-binding portion thereof, a chemotherapeutic agent, a drug agent, a radioactive agent, a cytotoxic agent, an antibiotic, a small molecule, a nucleic acid, or a polypeptide.
 48. The method of claim 1, wherein the diagnostic agent is a fluorophore, a fluorescent dye, a radionuclide, or an enzyme.
 49. The method of claim 1, wherein the microbial transglutaminase is a Streptomyces mobaraensis microbial transglutaminase. 50.-57. (canceled)
 58. The method of claim 1, wherein the ratio of the functional agent to immunoglobulin, or antigen-binding portion thereof, is 1:1 to 100:1 or 1:1 to 200:1.
 59. A conjugated immunoglobulin comprising an immunoglobulin or an antigen-binding portion thereof, and a functional agent, wherein a) the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, b) the functional agent comprises an acyl donor substrate, wherein the acyl donor substrate comprises a glutamine residue, and c) the functional agent is a therapeutic agent or a diagnostic agent, wherein the engineered lysine residue of the immunoglobulin, or antigen-binding portion thereof, is conjugated to the glutamine residue of the acyl donor substrate of the functional agent.
 60. A conjugated immunoglobulin comprising an immunoglobulin, or antigen-binding portion thereof, and a functional agent, wherein a) the immunoglobulin, or antigen-binding portion thereof, comprises an engineered lysine residue, wherein the engineered lysine residue is a lysine residue insertion or a natural amino acid residue which has been mutated to a lysine residue, b) the engineered lysine residue is conjugated to a glutamine residue on an acyl donor substrate, wherein the acyl donor substrate further comprises a reactive group, c) the reactive group is conjugated to a functional agent, wherein the functional agent is a therapeutic agent or a diagnostic agent. 61.-115. (canceled)
 116. A pharmaceutical composition comprising the conjugated immunoglobulin of claim 59, and a pharmaceutically acceptable carrier.
 117. A conjugated immunoglobulin produced by the method of claim
 1. 